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HEXRY  I.  FLEISSIG 


BACTERIOLOGY 


PITFIELD 


HENRY  I.  FLEISSIG 


From  The  Southern  Clinic. 

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BLAKISTON'S      ?QUIZ-COMPENDS? 

A  COMPEND 

ON 

BACTERIOLOGY 


INCLUDING  ANIMAL  PARASITES 
HENRY  I.  F-LSISSIO 


BY 

ROBERT  L.' PITFIELD,  M.  D. 

PATHOLOGIST   TO  THE   GERMANTOWN   HOSPITAL;   LATE   DEMONSTRATOR  OF 
BACTERIOLOGY  AT   THE  MEDICO-CHIRURGICAL   COLLEGE,   PHILA- 
DELPHIA; VISITING  PHYSICIAN  TO  ST.   TIMOTHY'S   HOS- 
PITAL AND  CHESTNUT  HILL  HOSPITAL,   PHILA. 


SECOND  EDITION 


WITH  4  PLATES  AND  85  OTHER  ILLUSTRATIONS 

P(.6 

PHILADELPHIA 

P.   BLAKISTON'S   SON   &   CO. 

1012  WALNUT  STREET 
1914 


Copyright,  1913,  By  P.  Blakiston's  Son  &  Co. 


THE. MAPLE. PRESO.YORK.PA 


PREFACE. 

This  little  book  was  designed  by  the  writer  to  serve  the  needs 
of  the  medical  student  preparing  for  examination,  and  for  the 
practitioner  of  medicine  who  desires  to  acquaint  himself  with  the 
principal  facts  of  the  rapidly  growing  science  of  bacteriology.  An 
effort  has  been  made  to  reduce  the  subject  matter  to  as  concrete 
a  form  as  possible. 

While  the  literature  of  the  subject  of  immunity  is  as  vast  almost 
as  the  rest  of  bacteriology,  yet  it  is  hoped  that  the  chapter  in  this 
book  on  immunity  gives  in  outline  the  essential  accepted  teachings 
on  the  subject. 

Minute  details  of  cultures  and  technic  are  not  given.  They  must 
be  sought  for  in  books  on  descriptive  bacteriology. 

The  author  has  drawn  very  freely  from  many  standard  text- 
books. Many  illustrations  are  from  Kalle  &  Wasser-mann's 
Atlas,  Williams,  McFarland,  Tyson's  Practice  and  Abbott. 

The  writer's  best  thanks  are  tendered  to  Dr.  Herbert  Fox  of  the 
University  of  Pennsylvania  (Pepper  Laboratory)  to  whom  entire 
credit  is  due  for  the  chapters  on  filterable  viruses;  the  rearrange- 
ment of  chapter,  and  the  new  matter  that  has  been  added 
throughout  the  book. 

To  the  firm  of  P.  Blakiston's  Son  &  Co.  the  writer  is  indebted 
for  valuable  aid. 

Robert.  L.  Pitfifxd. 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/compendbaGteriaOOpitfrich 


HENUVI.ILEISSIG 

TABLE  OF  CONTENTS 


Pagb 
CHAPTER  I. 

The  Classification,  Morphology,  and  the  Biology  of  Bacteria i 

CHAPTER  II. 
Products  of  Bacterial  Energy 21 

CHAPTER  III. 
Infection 27 

CHAPTER  IV. 
Immunity .• 41 

CHAPTER  V. 
Study  of  Bacteria 83 

CHAPTER  VI. 
Bacteriological  Laboratory  Technic 96 

CHAPTER  VII. 
Antiseptics  and  Disinfectants 120 

CHAPTER  VIII. 
Bacteria 127 

CHAPTER  IX. 
Animal  Parasites 211 

CHAPTER  X. 

The  Filterable  Viruses 234 

CHAPTER  XI. 
Bacteriology  of  Water,  Soil,  Air  and  Milk 261 

Index 271 

vii 


HENRY  I.  FLEISSIO 


COMPEND   OF  BACTERIOLOGY. 


CHAPTER  1. 


THE  CLASSIFICATION,  MORPHOLOGY,  AND  THE 
BIOLOGY  OF  BACTERIA. 

X  BACTERIA  (fission  fungi  or  schizomycetes)  may  be  defined 
as  very  minute,  unicellular  vegetable  organisms,  almost  always 
devoid  of  chlorophyll, ^nd  generally  unbranchedjthat  reproduce 
themselves  asexually  by  means  of  direct  division  or  fission,  spores^ 
or  gonidia.  They  are  allied  closely  on  the  one  hand  to  the  higher 
fungi,  such  as  the  moulds,  and  on  the  other  to  the  algae.  Many  forms 
in  one  phase  of  development  closely  resemble  members  of  other 
groups,  and  it  has  always  been  difficult  to  classify  them.  "  Various 
botanical  classifications  have  been  employed  by  different  bacteriol- 
ogists. The  following  one  is  based  somewhat  upon  Migula'Sy 
and  that  adopted  hy  Lehmann  and  Neumann,  which  was  compiled 
from  the  systems  of  FlUgge,  Fischer,   Loffler,  and  Migula, 

CLASSIFICATION  .^Bacteria  may  be  conveniently  divided  into 
six  families,  according  to  their  morphology  or  shape.  j| 

I.  "Jt^COCCACEiE.^Sphencal  or  spheroidal  bacteriaX  (Globular 
in  free  state  but  usually  seen  with  one  axis  slightly  larger. 
They  do  not  have  parallel  sides  like  the  bacilJi^Xro  mul- 
tiply, the  cell  divides  into  halves,  quarters,  or  eighths, 
each  of  which  grow  again  into  perfect  spheres. -^pndos- 
pores  and  flagella  are  very  rare.  (Lehmann  and  Neumann.) 
If  mobile  they  are  called  Planococcus  or  Planosarcina. 


2  BACTERIA 

^  (a)  Streptococcus. — Cells  that  divide  in  one  direction  only 

and  grow  in  chains.   "^ 
^  (b)  Micrococcus. — Cells  that  divide  in  two  directions,  or 

irregularly;   with    this   group   staphylococcus    may   be 

classed.     Also  tetrads,  which  form  into  fours  by  division 

in  two  directions.  ■^ 
^  (c)  Sarcina.- — Cells  that  divide  in  three  directions  so  that 

balelike  packages,  or  blocks  of  eight  are  formed.JV-At 

least  one  variety  (Sarcina  agilis)  is  motile,  having  flagella. 

Plates  of  cocci,  one  thick  in  the  plane,  are  called  '^meris- 

mopedia." 

II.teACTERIACE^.— Rod  bacteria  are  straight  or  slightly 
curved.  j(  Each  cell  is  from  two  to  six  times  as  long  as  broad. 
Division  takes  place  in  one  direction  only,  and  at  right  angles 
to  the  long  axis.  Spores  may  be  produced  or  may  not. 
They  may  have  flagella,  or  may  not. 

(a)  Bacterium.  Neumann — Have  no  endospores.  Migula 
— no  flagella. 

(b)  Bacillus.  Neumann — Have  endospores,  and  often 
grow  in  long  threads.  Migula — Flagella  present  at  any 
part  of  cell. 

(c)  Pseudomonas.  Have  endospores  very  rarely.  Fla- 
gella only  at  ends. 

III.  )LSPIRILLACEiE.— Spiral  bacteria*  Unicellular,  more  or 
less  elongated.  Twisted  more  or  less  like  a  corkscrew. 
Cells  are  sometimes  united  in  short  chains. j(^  Generally 
very  motile.     Spores  are  known  in  two  varieties  only. 

(a)  Spirosoma  rigidly  bent.     No  flagella. 

(b)  Vibrio  or  Microspira.  Cells  that  are  rigidly  bent  like 
a  comma,  and  have  always  one,  occasionally  two  polar 
flagella. 

(c)  Spirillum.  Are  long  and  spiral,  like  a  corkscrew,  are 
rigid,  and  have  a  bunch  of  polar  flagella. 


CHLAMYDOBACTERIACE^  3 

(d)  Spirochaeta.  Cells  with  long  flexible  spiral  threads, 
without  flagella.  They  move  by  means  of  an  undulating 
membrane.  These  have  been  thought  to  belong  to  the 
bacteria  but  since  we  now  know  that  most  of  them  move 
by  an  undulating  membrane,  they  should  be  classified 
with  the  protozoa. 

IV.  MYCOBACTERIACEiE.— Cells  as  short  or  long  filaments, 

which  are  often  cylindrical,  clavate  (club  shaped),  cuneate 
or  irregular  in  outline,  and  display  true  or  false  branching. 
Spores  are  not  formed,  but  gonidia  are.  They  have  no 
flagella,  and  division  takes  place  at  right  angles  to  the  long 
axis.  There  is  no  surrounding  sheath  as  in  the  next 
family  (V). 

(a)  Mycobacterium.  Cells  are  short  cylindrical  rods,  some- 
times wedge-like,  bent,  or  Y  shaped:  long  and  filamentous. 
They  exhibit  true  branching,  and  perhaps  produce  coccoid 
elements  and  gonidia,  but  no  flagella.  The  Corynebac- 
terium  of  Lehmann  and  Neumann  belongs  to  this  group. 
Many  are  acid  fast. 

(b)  Streptothrix  or  Actinomyces  {ray  fungus)  are  long 
mycelial  threads,  that  radiate  in  indian-club,  or  loop-like 
forms,  with  true  branching  and  delicate  sheaths,  devoid  of 
gonidia  and  flagella.  Growth  coherent,  mould-like  and 
dry.  Often  powdery  on  the  surface  in  culture  media. 
Not  acid  proof  or  acid  fast,  and  frequently  emit  a  musty 

V.  CHLAMYDOBACTERIACE.^.— Sheathed  bacteria.     Cells 

are  characterized  by  an  enveloping  sheath  about  branched 
and  unbranched  threads.  Division  takes  place  at  right 
angles  to  the  long  axis  of  the  cells. 

(a)  Cladothrix  are  distinguished  by  false  dichotomous 
branching.     Multiplication  is  affected  by  separation  of 


4  BACTERIA 

whole  branches,  and  by  swarm  spores  or  motile  gonidia 
having  flagella. 

(b)  Crenothrix.  Filaments  are  fixed  to  a  nutrient  base. 
Are  usually  thinner  at  the  base  than  at  the  apex,  formed 
of  unbranched  threads  that  divide  in  three  directions  of 
space,  and  produce  in  the  end  two  kinds  of  gonidia, 
probably  of  bisexual  nature. 

(c)  Phragmidiothrix.  Cells  are  first  united  into  un- 
branched threads  by  means  of  delicate  sheaths,  branching 
threads  are  then  formed.  Division  takes  place  in  three 
directions  of  space,  producing  sarcina-like  groups  of  goni- 
dia, which,  when  free,  are  spherical. 

(d)  Thiothrix.  Are  unbranched  cells,  sheathed,  without 
flagella,  divided  only  in  one  direction,  and  contain  sulphur 
granules. 

VI.     BEGGIATOACEiE.     Cells  united  to  form  threads  that  are 
not  sheathed:  have  scarcely  visible  septa;  divide  in  one  direc- 
tion, and  motile  only  by  an  undulating  membrane,  not  by 
flagella. 
(a)  Beggiatoa.     Cells  containing  sulphur  granules. 

^  Bacteria  may  furthermore  be  classified  according  to  their  biolog- 
ical characteristics,  which  may  be  wonderfully  diiferent.  The  ulti- 
mate differentiation  of  one  species  from  another  depends  not  only 
on  the  morphology,  which  may  be  precisely  similar,  but  on  its  biolog- 
ical behavior  in  culture  media  and  in  the  tissues  of  animals  under 
identical  conditions.  Again,  different  individuals  of  a  given  species 
may  vary  extraordinarily  one  from  another  in  form  and  size,  yet  the 
chemical  behavior  is  invariably  the  same.  Hence  it  is  only  by  obser- 
vation of  the  development  of  bacteria  in  culture  media,  and  the  re- 
actions produced  in  it,  and  in  the  bodies  of  experiment  animals,  that 
we  can  identify  them  positively  from  others  of  a  foreign  species.  No 
bacteriologist  is  able  by  a  simple  microscopical  examination  of  a 
given  bacterium,  to  identify  it  absolutely  at  all  times.  -^ 


CLASSIFICATION 


^  Bacteria  that  are  globular  in  form  are  called  cocci.  J( 
i/L   Cocci  that  divide  in  one  direction  of  space  and  grow  in  chains  are 
called  streptococci.  /  (Fig.  i.) 

^  Cocci  that  divide  irregularly  and  form  pairs  or  fours,  or  irregular 
groups,  are  called  micrococci.     Those  of  this  class  that  form  pairs 


Fig.  I. — Large  and  very  large  streptococci.     (Kolle  and  Wassermann.) 

are  frequently  called  diplococci.  When  they  form  fours  by  division 
in  two  directions,  they  are  called  tetrads.  But  when  they  divide 
irregularly  and  form  masses  resembling  bunches  of  grapes,  they  are 
spoken  of  as  staphylococci.  X  (Fig.  2.) 


Fig.  2. — Staphylococci.    Streptococci.     Diplococci.    Tetrads.         Sarcinae.      (.Williams.) 

\  Cocci  that  divide  in  three  directions  are  called  sarcince.  One 
single  coccus,  by  division  in  three  directions,  forms  cubes  of  eight 
or  more,  each  of  which  becomes  globular  and  equal  in  size  to  the 
parent.    4 


O  BACTERIA 

^  Motile  micrococci  are  those  that  divide  in  two  directions  of  space, 
and  have  flagella.  fsThey  are  known  as  planococci. 

Micrococci   that  divide  in  three  directions,  and  are  motile,  are 
C2i\\ed  piano sarcifhcd,     (Fig.  3.) 

fvBacteria  that  resemble  straight  rods  are  called  bacilli.  These 
may  be  short  and  thick,  or  long  and  thread-like;  are  never  curved, 
but  may  be  slightly  bent.  ^ 


Fig.  3. — ^Planosarcina  ureae,  showing  very  long  flagella. 
(Kolle  and  Wassermann.) 

^Bacilli  may  grow  singly  or  in  chains;  may  be  flagellated;  contain 
spores  and  gonidia;  or,  may  be  devoid  of  flagella.  "j^ 

Members  of  the  spirillaceae  that  resemble  a  curved  rod,  or  are 
comma  shaped,  are  known  as  vibrios.  (Fig.  4.)  Those  of  the 
same  family  that  resemble  a  corkscrew,  are  called  spirilla.  (Fig.  5.) 
When  they  are  like  long  spiral  threads  they  are  called  spirochcetce . 

Any  of  these  different  members  of  the  family  of  Spirillaceae  may 
grow  in 'chains. 

In  clinical  medicine  it  is  common  to  speak  of  the  streptococcus 
lanceolatus  as  the  pneumococcus.  As  the  organism  appears  in  the 
diseased  lung,  or  in  the  sputum,  one  diameter  of  the  coccus  is  invaria- 
bly longer  than  another,  and  the  rule  of  equal  diameters  cannot  be 


•     CLASSIFICATION  7 

applied  to  it.  But  in  culture  mediae  the  organism  resembles  a  true 
coccus,  being  globular  and  growing  in  chains.  It  is  then  called 
the  Streptococcus  lanceolatus.  It  is  common  also  to  speak  of 
members  of  the  family  of  Mycohacteriacece.  as  bacilli,  as  they  are 
more  commonly  met  with  in  this  form  in  clinical  examinations,  and 
in  cultures.  Hence,  we  frequently  hear  of  the  bacillus  of  tubercu- 
losis, and  not  the  Mycobacterium  tuberculosis. 

Among  the  higher  bacteria,  the  differentiation  of  those  belonging 
to  the  sheathed  group,  or  Chlamydobacteriacece,  is  difficult,  as  it 
depends  largely  upon  the  formation  of  the  false  branching  and  the 
gonidia.  When  bacteria  exhibit  many,  or  various  forms,  in  the 
same  culture,  as  does  the  typhoid  bacillus,  we  speak  of  it  as  pleo- 


FiG.  4. — Cholera  vibrios. 
(Greene's  Medical  Diagnosis.) 


Fig.  5. — Spirillum  relapsing  fever. 
(Greene's  Medical  Diagnosis.) 


morphic,  or  pleomorphism.  To  elucidate:  Man  is  pleomorphic, 
because  among  adult  individuals  some  are  tall  or  short,  fat  or  thin. 
-^  Involution  or  Degeneration  forms.  When  the  best  or  optimum 
conditions  for  bacterial  life  (see  page  17)  are  not  found,  bacteria 
present  appearances  quite  different  from  those  of  the  young,  active 
or  perfect  adult  type.  These  are  called  involution  or  degeneration 
forms.  For  example:  the  diphtheria  bacillus  under  good  conditions 
for  life  is  a  straight  or  slightly  bent  rod  staining  in  a  granular  man- 
ner. If  living  under  unsuitable  conditions  it  becomes  quite  short, 
and  stains  solidly.  Ag'ain  bacilli  that  are^  accustomed  to  appear  as 
short  elements  may  grow  to  long  threads  without  dividing,  or  swell 
into  unrecognizable  form.     / 


8  BACTERIA 

y  To  measure  bacteria,  we  use  the  thousandth  part  of  a  millimeter, 
called  the  micromillimeter,  or  micron,  as  the  unit?*  (xhe  Greek  let- 
ter //  is  the  symbol  for  this  unit)Av\  micron  is  about  ywUu  of  an 
inch,  yet  a  bacterium  one  fi  long,  and  a  half  ft  in  width,  is  very 
large  in  comparison  to  some  things  that  scientists  measure,  such 
as  the  thickness  of  oil  films,  soap  bubbles,  or  light- wave  lengths,  in 
which  the  unit  is  a  micro-micron,  and  is  symbolized  by  n/i.  The 
shortest  light-wave  lengths  are  about  400  jujut,  or  .4  //,  while  chromatic 
threads  in  cells  of  bacteria  are  often  100  juji  in  width.  Then 
again  there  are  many  things  smaller  than  these  threads.  The  thin- 
nest part  of  a  bursting  soap  bubble  is  but  7  /jl/i 
in  thickness.  There  are  certain  infectious 
agents  that  are  submicroscopic;  that  is,  in- 
visible even  by  the  aid  of  Siedentopf's 
ultraviolet  microscope,  which  shows  objects 
smaller  by  half  a  light-wave  length  (.2  pL/x). 
^  The  structure  of  the  bacterial  cell  is  very 
PiQ  6.— Diplococci  simple.  It  consists  of  (i)  a  central  nuclear 
showing  capsules  \^Q^y  ^iij^h  can  be  stained  like  the  nuclei  of 
(Greene's    Medical        ,  1.1,,.,  , 

Diagnosis.)  other  vegetable  and  animal  cells,  with  nuclear 

or    basic    stains,    such    as   haematoxylin,    or 
methylene-blue.  \ 

<  (2)  A  cytoplasm,  or  protoplasmic  substance  generally  very  thin.^l 
J^(3)  A  cell  wall,  more  or  less  thick,  that  stains  with  difficulty. 
(Fig.  6.) 

'Lin  the  nucleus  we  often  see  metachromatic  bodies,  called  the 
Babes-Ernst  granules,  and  unstained  spaces  called  vacuoles ,  both  of 
which  are  common  to  many  bacteria.  These  are  both  probably 
due  to  ingested  food  or  fluid,  -f^ 

>  Through  the  cell  wall  the  food  of  the  bacterium  passes  by  osmosis.  -J- 
4NThe  cell  wall  of  certain  organisms,  for  example  the  pneumococcus, 
undergoes  a  change  whereby  a  mucilaginous  or  gelatinous  capsule 
is  formed  outside  the  cell  wall.     Its  use  is  not  known.     The  cell 
wall  is  generally  the  first  portion  of  the  cell  to  be  attacked  by 


CLASSIFICATION  9 

certain  specific  substances  (ferment)  found  in  the  blood  of  immu- 
nized animals,  called  hacteriolysins  and  agglutinins^  Where 
great  masses  of  bacteria  are  clumped  in  excessive  mucilaginous 
material  we  speak  of  this  condition  as  zooglea.  (Fig.  7.) 
^  We  sometimes  find,  as  a  prolongation  of  the  cell  wall,  filamentous 
organs  of  locomotion  known  as  flagella.     Not  all  bacteria  possess 


Fig.  7. — Zooglia  formation.     (Leuconostoc.)     (Kolle  and  Wassermann.) 

these^ut  those  that  do,  are  called  trichohaderia.  Those  that  have 
not/flagellaSare  called  gymnobacteria.  Trichobacteria  are  classified 
accoi^iw^^  the  number  and  location  of  the  flagella.  When  they 
have  one  flagellum  we  call  them  monotrichous  bacteria,  and  amphi- 
trichous  when  there  are  two  flagella,  one  at  each  pole.  (Fig.  8.) 
When  the  cell  is  surrounded  by  flagella,  it  is  known  as  a  peritrichous 
bacterium,  and  lophotrichous  when  the  flagella  are  arranged  in  tufts 
of  two  or  more.  These  are  simple  adjectives  and  not  now  used 
as  terms  of  classification.  The  tetanus  bacillus  is  an  example  of  a 
peritrichous  organism,  while  the  bacillus  of  green  pus  is  called 
monotrichous,  because  of  its  single  flagellum.  (Fig.  9.) 
♦  Flagella  are  not  pseudopods,  but  distinct  organs  of  locomotion. 
In  certain  bacteria  of  the  Beggiatoa,  locomotion  is  accomplished 


lO  BACTERIA 

by  a  peculiar  amoeboid  motion,  or  by  an  undulating  membrane. 
On  looKng  at  bacteria  known  to  have  no  powers  of  voluntary 
motion,  they  are  seen  to  oscillate,  tremble  or  move  slightly.  Sus- 
pensions of  india-ink  in  water  are  seen  to  do  the  same  thing,  as  are 
other  inanimate  suspensions.  This  molecular  movement  is  known 
as  the  Brownian  motion.     There  are  bacteria  that  are  considered 


Fig.  8. — Spirillum  undula  with  polar  Fig.  9  — Bacillus  prolius  vul- 

flagella.    (Kolle  and  Wassermann .)  garis,  showing  peritrichous  fla- 

gella.  (Kolle  and  Wassermann.) 

non-motile,  on  which  it  is  possible  to  demonstrate  flagella.  By 
ordinary  staining  methods,  and  in  preparations  of  living  bacteria 
known  to  be  flagellated,  these  organs  of  locomotion  cannot  be  seen, 
as  a  rule.  Occasionally,  however,  one  may  be  seen  under  either 
condition.  Generally,  strong  solutions  of  aniline  dyes,  to  which 
powerful  mordants  have  been  added,  are  necessary  to  stain  the 
capsule  of  bacteria  and  the  attached  flagella.  The  motion  or 
bacteria  varies  from  a  simple  rotatory,  on  one  axis,  to  a  swinging, 


REPRODUCTION  II 

shaking,  boring  or  serpentine  action.  The  location  of  the  flagella 
has  some  influence  upon  their  behavior.  Flagella  may  be  broken 
off  from  the  cell  body  by  agitation.  They  are  then  clumped  by 
agglutinating  sera. 

Flagella  may  have  other  functions  than  locomotion.  It  is  possible 
that  they  may  serve  as  organs  for  the  absorption  of  nourishment 
from  the  surrounding  media.  The  presence  of  very  long  or  very 
numerous  flagella  does  not  necessarily  presage  very  active  motion. 
At  times,  under  certain  conditions,  an  organism  ordinarily  motile 
and  flagellated  will  appear  immobile  and  non-flagellated  {Lehmann 
and  Zierler),  but  this  is  rare.  Certain  flagella  have  in  their  contin- 
uity little  round  granules,  or  bodies,  which  apparently  have  nothing 
to  do  with  the  functions  of  locomotion,  but  may  have  something 
to  do  with  the  nutrition  of  the  cell.  The  test  of  motility  of  a 
bacterium  is  to  see  it  progress  by  itself  completely  across  the  field 
of  the  microscope. 

t  REPRODUCTION.— The  process  of  direct  cell  division  is  the 
commonest  way  by  which  bacteria  multiply.  Hence  the  name  of 
fission  fungi.  The  ways  of  reproduction  of  the  bacteria  high  in  the 
scale  are  by  direct  division,  branching,  and  by  means  of  spores, 

^nd  by  other  granules  called  gonidia.J  The  spores  appearing  in  the 
lower  bacteria,  bacilli  for  example,  are  not  reproduction  forms  but 
states  of  high  resistance. 

The  process  of  cell  division  or  binary  division  is  very  simple,  and 
may  be  a  matter  of  twenty  minutes,  or  as  long  as  six  hours.  Divi- 
sion is  almost  always  across  the  cell  in  the  direction  of  the  short  axis , 
though  it  may  in  some  bacteria  be  in  a  direction  parallel  to  the  long 
axis,  but  this  is  uncommon. 

By  means  of  the  hanging  drop  or  the  block  culture  method,  on 
an  inverted  cover -glass,  the  process  may  be  observed  easily.  The 
phenomena  of  division  begin  by  an  elongation  of  the  cell,  soon  fol- 
lowed by  a  constriction  or  pinching  in  of  the  cell  on  both  sides,  at  an 
equatorial  point.  The  process  begins  to  be  apparent  in  the  cell 
wall  and  extends  inward,  u 


12  BACTERIA 

Division  may  occur  in  one,  two,  or  three  directions,  or  planes. 

By  cell  division  bacteria  multiply  by  geometrical  progression. 
One  cell  at  the  end  of  an  hour  becomes  two,  and  at  the  end  of  a 
second  hour  these  two  become  four;  at  the  end  of  another  hour  these 
four  become  eight;  after  twenty-four  hours  they  may  number  many 
millions. 

It  is  well  that  the  food  supply  soon  gives  out  and  that  the  products 
of  bacterial  metabolism,  such  as  acids  and  ferments,  inhibit  their 
growth.  By  this  rapid  bacterial  multiplication,  carcasses  of  animals 
are  disintegrated  and  the  higher  nitrogenous  compounds  are  re- 
duced to  simple  gases  that  are  quickly  dissipated  in  the  air. 
•k  SPORULATION.— Sporulation  is  of  two  kinds:  the  first  and 
most  important  for  hygiene  is  that  into  which  some  pathogenic 
bacteria  go  when  they  meet  unfavorable  conditions  and  it  affords 


Fig.  io. — The  formation  of    Fig.   ii. — Spores  and  their  location  in  bacteria  J 
spores.       (After  Fischer  from  cells.     (After  Frost  and  McCampbell.) 

Frost  and  McCampbell.) 

protection  against  all  but  the  most  vigorous  disinfection;  the  second 
kind  is  a  specialized  function  of  the  higher  bacteria  and  moulds  by 
which  reproduction  occurs.  In  the  latter  case  it  is  not  impossible 
that  some  sexual  specialization  occurs.  The  first  mentioned 
are  called  Endospores. 

Vegetative  sporulation  corresponds  to  the  flowering  of  the  higher 
plants,  and  is  observed  under  the  most  favorable  vital  conditions. 


SPORULATION  13 

Endospores  are  produced  under  stress  of  circumstances,  when 
certain  agencies' or  conditions,  such  as  absence  of  food,  drying,  and 
heat,  threaten  the  extinguishment  of  the  organism.  Spores  are 
bright,  shining,  oval,  or  round  bodies,  which  do  not  take  aniline 
dyes  readily,  and  which,  when  they  are  stained,  retain  the  color 
more  tenaciously  than  the  adult  cells.  They  resist  heat,  often  with- 
standing a  temperature  of  150°  C.  dry  heat  for  an  hour.  Steam 
under  pressure  at  a  temperature  of  150°  C.  will  invariably  kill  them 
after  a  short  exposure. 

Spores  are  situated  either  in  the  ends  of  the  adult  organism 
(polar)  or  in  the  middle  (equatorial),  and  the  spore  is  discharged 
(sporulation)  either  from  the  end  or  through  the  side. 

(1)    Q    f,    I  fe      ^^    I  k^^ 

0  1^ 


0   0   0 


5  8 


Fig.  12. — Spore  germination,  a,  direct  conversion  of  a  spore  into  a  bacillus 
without  the  shedding  of  a  spore- wall  {B.  leptosporus) ;  h,  polar  germination  of 
Bad.  anthracis;  c,  epuatorial  germination  of  B.  suhtilis;  d,  same  of  B.  mega- 
terium;  e,  same  with  "horse-shoe"  presentation.     (After  Novy.) 

The  spore  is  developed  in  the  bacterial  cell  as  follows:  If  the 
organism  is  a  mobile  one  it  becomes  quiet  before  sporulation,  during 
which  the  flagella  are  retained.  The  diameter  becomes  greater  in 
one  portion  of  the  cell,  and  dust-like  particles  appear,  then  a  bright 
spot;  a  capsule  then  forms,  the  spore  escapes,  and  the  parent  cell 
dies,  i 

^  Certain  spore  bearing  bacteria  grown  for  a  week  at  42°  C.  lose  the 
power  to  form  spores,  likewise  their  progeny.  As  a  rule  the  anthrax 
bacillus  does  not  form  spores  in  the  bodies  of  animals.  Free  oxygen 
'is  required  for  sporulation  by  some  bacteria.  Qiie_spore  only  i<;  pio- 
duced  by  an  adult  cell.     Some  forms  of  bacteria  can  be  diffejeiit^itQd 


14  BACTERIA 

from  each  other  only  by  the  way  in  which  they  sporulate,  whether 
from  the  poles  or  the  equator.  K 

V  The  BacterlacecB  are  the  prominent  spore  producers.  Certain 
round  bodies  found  in  bacteria  of  low  thermal  death-point,  are 
called  by  Heuppe  arthrospores.  It  is  believed  that  they  are  without 
significance.  A  high  thermal  death-point  in  bacteria  indicates 
that   the  organism   produces   spores.     Arthrospores  are  common 


"^t:. 


Fig.  13. — Capsules.     Bad.  pneumonicB  (Friedlander) .      (After  Weichselbaum 
from  Frost  and  McCampbell.) 


among  the  micrococci  and  may  be  associated  with  capsule  forma- 
tion and  cell  enlargement.  The  whole  cell  may  stain  more 
intensely.  They  are  also  to  be  sought  among  the  Strep tothrix 
genus.  > 

"fiv  Spores  resist  chemicals  for  a  long  period,  and  withstand  drying, 
even  in  lime  plaster,  for  years.     It  is  believed  that  the  thick  capsule 
enables  them  to  resist  these  deleterious  agents.^ 
^  Sporulation  is  more  apt  to  occur  under  poor  nutritive  conditions.  Y 
The  anthrax  bacillus  thrives  at  13°  C.  but  cannot  sporulate  below 


SPORULATION 


15 


18°  C.     Anthrax  spores  have  been  known  to  resist  the  germicidal 
action  of  a  5  percent  carbolic  acid  solution  for  forty  days. 

Babes-Ernst  granules,  or  polar  bodies  are  found  in  certain 
bacteria  (Mycobacteriaceae,  etc.)  after  staining  with  special  basic 
stains.  In  the  complex  forms  of  bacteria,  they  evidently  have  an 
important  role  in  reproduction.  The  presence  of  such  bodies  in  the 
poles  of  diphtheria  bacilli  facilitates  the  recognition  of  these  organ- 
isms.    (Fig.  14.) 


Fig.  14. — ^Pest  bacilli  showing  capsules.     (Kolle  and  Wassermann.) 

i  Capsules.  Certain  well  known  pathogenic  bacteria  have  thick 
well  marked  capsules.  The  pneumococcus,  pneumobacillus,  and 
Bacillus  aero  genes  capsulatus,  are  well  known  examples  of  such 
capsulated  organisms.  The  capsule  is  not  always  constant.  It 
often  disappears  when  the  organism  is  grown  in  culture  media.  JC 
(Fjg^^ 

The^hi^her  bacieria/2Lre  those  from  the  Mycohacteriacece  up  to  the 
[easts  and  moulds./  They  are  higher  than  the  Bacteriacece  because 
they  tend  to  form  truly  or  falsely  branching  filaments  and  specialized 
segments,  gonidia,  which  may  behave  as  sex  organs.  Few  of  them 
are  pathogenic,  except  in  the  genera  Mycobacterium  and  Strepto- 


1 6  BACTERIA 

thrix.  To  the  former  belongs  the  diphtheria  and  tubercle  bacillus, 
both  of  which  are  said  to  have  branching  involution  forms,  while  to 
the  latter  belong  the  organisms  of  actinomycosis  and  Madura  foot. 
The  Chlamydohacteriacece.  and  Beggiatoa  are  Saprophytes.  These 
require  special  technique  for  their  laboratory  culture. 
Y^The  Yeasts  or  Blastomycetes  or  budding  fungi  are  next  in  order. 
They  consist  of  sharply  and  doubly  outlined,  refractive,  oval  bodies 
which  may  grow  out  into  short  stalklcalled  mycelia.  ^They  grow 
well  in  the  laboratory  and  may  produce  pig- 
ments. They  are  much  larger  than  the  bac- 
teria (ia-25  /x  long).  They  multiply  by  bud- 
ding with  a  separation  and  removed  growth 
of  the  young  form.  They  may  produce  a  local 
or  general  infection  in  man.  Blastomycosis. 
They  are  used  in  beer  making.  The  com- 
PiQ_  15, B  a  c  i  1 1  i    monest  genus  is  Saccharomyces. 

showing    capsules         xhe  Moulds  or  .Hyphomycetes  represent  the 
(Greene's    Medical  ,  .  <r  ^^~V^  1  ,  r^^ 

Diagnosis.)  next  highest  group  of  the  plant  algae.     They 

are  characterized  by  a  greater  prominence  of 
the  mycelium  over  simple  segments  or  bodies.  They  are  wide- 
spread in  nature  and  many  are  pathogenic.  They  multiply  by 
segmentation  of  the  mycelia  into  gonidia  or  by  the  development  of 
special  spore  masses  called  sporangia.  Further  refinements  of  the 
spores  into  sexual  elements  is  known.  They  are  chiefly  of  interest 
to  the  physician  on  account  of  the  skin  diseases  that  they  occasion. 

THE  CHEMICAL  COMPOSITION  OF  BACTERIA. 

^A  Bodies^  of  bacteria  contain_water,_saItv .certain  albumins,  and 
bodies  that  may  be  extracted  with  ethgrX  Among  the  latter  are 
lecithin,  cholesterin,  and  triolein.^  In  the  tubercle  bacillus,  fatty 
acids  and  wax  have  been  found.  In  others,  xanthin  bases,  cellulose, 
starch,  chitin,  iron  salts,  and  sulphur  grains  have  been  discovered. 
The  essential  albumin  of  the  cell-body  is  highly  nitrogenous  and  is 
called  mycoprotein.     The  salts  in  the  ash  are  mostly  composed  of 


BIOLOGICAL  CONDITIONS  1 7 

various  phosphates.  Intracellular  toxins  in  combination  with 
the  cytoplasm  are  found  in  certain  groups  of  bacteria,  e.g.,  B, 
typhosus.  pC 

BIOLOGICAL  CONDITIONS. 

y(  Bacteria  are  arbitrarily  classed  either  as  parasites,  or  saprophytes^ 
They  may  be  so  dependent  upon  the  tissues  of  the  infected  organism 
as  to  be  a  strict  parasite  and*  incapable  of  growth  under  any  other 
con6.\iioit{M ycohact.  leprce) ,  or  they  may  be  capable  of  life  on  arti- 
ficial culture  media  (tubercle  bacillus),  or  of  life  in  the  body,  on 
culture  media,  or  in  the  soil  (B.  tetani).  fC 

j(  The  bacteria  of  the  soil,  water,  air,  etc.,  that  are  incapable  of 
successful  life  in  the  body  tissues  are  called  saprophytes,  j^ 
y(  Certain  biological  conditions  are  essential  for  the  growth  of  bac- 
teria: water,  oxygen,  carbon,   nitrogen,  and  salts   are   necessary. 
For  certain  parasitic  bacteria,  highly  complex  substances  are  indis- 
pensable: meat  albumins,    peptones,    milk,  egg    albumin,  blood 
serum,  and  sugars  are  the  ingredients  of  various  culture  media,  y 
)<.  The  chemical  reaction  of  such  media  is  important:  it  should  either 
be  faintly  acid  or  faintly  alkaline.     The  greatest  number  of  water 
bacteria  grow  in  media  that  are  slightly  acid,  while  diphtheria  prcjji^ 
duces  its  strongest  toxins  and  grows  best  in  alkaline  media.     Salt 
free  mkiia  is  required  for  a  number  of  pathogenic  bacteria,  e.g. 
the  Gonococcus,  B.  Leprae.^ 

)(  All  bacteria  require  for  their  growth  either  free  oxygen,  as  in  air, 
or  combined  oxygen,  as  in  albumin,  water,  etc.  Those  that  only 
grow  when  deprived  of  free  oxygen  are  known  as  obligate  anaerobes^ 
while  those  that  require  the  presence  of  oxygen  are  called  obligate 
aerobes^  Those  that  grow  under  either  conditions  are  namecfyoa*/- 
tative  anaerobes.  Free  oxygen  is  needed  for  spore  formation  by  cer- 
tain bacteria.  Anaerobes  obtain  oxygen  as  they  need  it  by  breaking 
up  their  food  stuffs,  y 

>y  Nutriment  is  most  important  for  the  growth  of  bacteria,  nitrog- 
enous compounds  (albumins)  particularly  being  required.     Simple 


l8  BACTERIA 

aquatic  forms  of  bacteria  can  live  and  grow  in  distilled  water.  The 
addition  of  the  various  sugars  is  of  advantage  in  the  cultivation  of 
many  bacteria,  and  glycerine  for  the  growth  of  some  members  of 
the  MycobacteriacecB.  Blood  serum  or  whole  blood  is  required  by 
some  pathogenic  organisms.  The  food  stuffs  must  be  in  a  form 
that  can  diffuse  through  the  cell  wall.  ^ 

^  The  temperature  of  the  medium  in  which  various  bacteria  grow 
is  most  important.  Bacterial  growth  is  possible  between  o°  C.  and 
70°  C,  some  varieties  thrive  at  the  one  extreme,  and  otH^rs  at  the 
other.    j( 

Psychrophilic  bacterid  are  those  that  grow  at  15°  C,  with  a 
maximum  of  30°  C.  and  a  minimum  of  0°  C.  Water  bacteria 
of  the  polar  seas  belong  to  this  group. 

Mesophilic  grow  best  at  37°  C. — the  temperature  of  the  body — 
and  thrive  from  10°  C.  (minimum)  to  45°  C.  (maximum).  All 
pathogenic  bacteria  belong  to  this  group. 

Thermophilic  (min.  temp.  40°  C,  max.  60-70°  C.)  are  most 
prolific  at  50-55°  C.  To  this  class  belong  bacteria  of  the  soil. 
All  of  this  class  are  spore  bearing. 

Darkness  favors  bacterial  growth. 
^Association  of  different  kinds  of  bacteria  is  of  some  importance 
in  their  growth  and  welfare.     When  thus  associated,  they  sometimes 
benefit  each  other.     Such  combination  is  called  symbiosis.  ]f^ 
V  Certain  anaerobic  bacteria  grow  i^^  the  presence  of  oxygen  if 
other  particular  varieties  of  aerobic  bacteria  are  present./ 
\  Attenuated  tetanus  bacilli  become  virulent  if  cultivated  with  Bac- 
terium vulgae.     Again,  complicated  chemical  changes,  as  the  decom- 
position of  nitrites  with  the  evolution  of  nitrogen  cannot  be  accom- 
plished by  certain  bacteria  severally,  but  jointly,   this  is  quickly 
brought  about. 'f' 

^Pfeiffer  has  shown  that  certain  chemical  substances  (foods,  albu- 
mins, etc.),  attract  bacteria  (positive  chemolaoHs),  while  other  sub- 
stances, as  turpentine,  repel  them  (negative  chemotaxis). 

Oxygen  repels  anaerobes  and  is  particularly  attractive  to  aerobes.  J[^ 


AGENTS  PREJUDICIAL  TO  BACTERIAL  LIFE  1 9 

FREE  AGENTS  PREJUDICIAL  TO  THE  LIFE  OF 
BACTERIA. 

High  temperatures  are  surely  germicidal:  60°  C.  coagulates 
mycoprotein  of  bacteria  and  other  common  albumins.  The  degree 
of  temperature  at  which  bacteria  are  killed  is  called  the  thermal 
d^atit^oint.  Most  vegetative  forms  die  after  a  short  exposure  at 
60°  C,  though  some  require  a  higher  temperature,  e.g.,  tubercle 
bacillus. 

>(  Spores  resist  boiling,  often  for  hours.  Spore-bearing  bacilli  from 
the  soil  often  survive  a  temperature  of  115°  C.  moist  heat  (steam), 
from  thirty  to  sixty  minutes.  Bacteria  resist  dry  heat  of  175°  C. 
from  five  to  ten  minutes.  < 

y  Cold  inhibits  bacteria;  destroys  some;  but  is  not  a  safe  germicidal 
agent,  as  typhoid  bacilli  have  been  isolated  from  melted  ice  in  which 
they  had  been  frozen  for  months.  .^ 

Ravenel  exposed  bacteria  to  the  extreme  cold  of  liquid  air  ( — 312° 
F.)  and  found  that  typhoid  bacilli  survived  an  exposure  of  sixty 
minutes;  diphtheria,  thirty  minutes,  and  anthrax  spores,  three  hours; 
during  this  exposure,  however,  many  were  destroyed.  In  each 
instance,  vegetative  forms  grew  after  the  exposure. 
/  Light  is  inimical  to  the  life  of  bacteria;  direct  sunlight  being  the 
most  germicidal,  as  it  destroys  some,  reduces  the  virulence  of  others, 
or  interferes  with  the  chromogenic  properties.  Typhoid,  cholera, 
diphtheria,  and  many  other  organisms  are  killed  after  an  hour  or 
two's  exposure  to  bright  sunlight.  The  ultraviolet  or  actinic  rays 
are  the  efficient  ones.  If  free  oxygen  is  excluded,  the  germicidal 
action  is  very  materially  reduced.  Sunlight  acting  on  culture  media 
(free  oxygen  and  water  being  present)  produces  after  ten  minutes, 
peroxide  of  hydrogen.  This  action  of  light  on  bacteria  has  been 
extensively  used,  notably  by  Hansen,  as  a  therapeutic  measure  for 
the  cure  of  bacterial  skin  diseases,  especially  lupus.  Diffuse  sun- 
light, electric  light,  Rcentgen-rays,  continuous  and  alternating 
currents  of  electricity,   are  also  more  or  less  germicidal.     Anti- 


20  BACTERIA 

septics,   such   as  metallic   salts,   formalin,   carbolic   acid,   cresol, 
mineral  acids,  and  essential  oils,  are  powerful  germicides;  some 
even  in  high  dilution.    / 
"7^  According  to  Koch,  absolute  alcohol,  glycerine,  distilled  water, 
and  concentrated  sodium  chloride  solution  do  not  affect  anthrax 
spores,  even  after  acting  on  them  for  months.     Halogen  elements 
(iodine,  bromine,  chlorine)  are  the  most  powerful  germicides.   >/ 
^  Free  acids  and  alkalies  must  be  very  strong  to  act  as  disinfectants. 
Excessive  amounts  of  sugar,  salt,  glycerine,  and  the  pyroHgneous 
acids  act  as  destroyers,  or  inhibitors  to  bacterial  growth  in  food  stuffs^ 
r^Metals  act  as  lethal  agents  in  the  presence  of  light  and  water,  by 
forming  metallic  peroxides,  which  either  destroy  the  vitality  of  bac- 
teria or  hinder  their  growth.     Silver,  zinc,  cadmium,  bismuth,  and 
copper,  have  this  action.     Consequently  silver  wire,  or  foil,  are 
used   in   surgery   because    of    their    anti-septic    action.     Metallic 
fillings  in  teeth  prevent  the  growth  of  bacteria  that  cause  caries, 
w    Certain  cells  in  the  bodies  of  animals  (leucocytes)  and  some  ele- 
ments of  the  blood  serum,  being  bactericidal,  are  a  powerful  means 
4Bf  internal  defense  against  infection,  j^ 

^  If  the  water  of  the  cytoplasm  of  bacterial  cells  is  dried  out,  the 
vitality  of  the  organism  suffers.  The  length  of  time  required  for 
drying  varies,  anthrax  spores  resisting  the  process  for  over  ten  years. 
Ancient  methods  of  preserving  foods  from  putrefying,  and  which  are 
still  in  vogue,  depend  upon  the  employment  of  some  of  these  agents, 
which  are  prejudicial  to  bacterial  life.  Meats  are  salted,  pickled, 
dried,  or  smoked.  Fruits  are  dried,  pickled,  or  immersed  in  strong 
saccharine  solution,  in  order  to  preserve  them  from  decay,  in  every 
instance,  the  absence  of  moisture,  the  excess  of  salt,  sugar,  or 
vinegar,  or  the  pyroligneous  acid  from  the  smoking,  prevents 
bacterial  growth,  and  consequently,  decay  of  the  food  stuff.  The 
products  of  bacterial  growths  often  inhibit,  or  destroy,  the  cells  that 
made  them,  as  well  as  other  bacteria.  B.  pyocyaneus  and  S. 
cholercEf  have  this  property  of  secreting  autolytic  ferments.y 


CHAPTER  II. 


PRODUCTS  OF  BACTERIAL  ENERGY. 

"iiC  According  to  their  chemical  activities,  bacteria  are  arbitrarily 
divided  into  the  following  classes: 

)(^    Photogens  Chromogens        Zymogens 

Saprogens  Aero  gens  Pathogens  y^ 

^MPhotogens  are  those  bacteria  of  the  sea,  putrefying  flesh,  and 
damp  rotten  wood,  that  produce  a  faint  phosphorescence.  >^ 
/>  Chromogens  are  bacteria  that  produce  colors  as  they  grow, 
notable  among  which  may  be  mentioned  the  Staphylococcus  aureus^ 
that  are  golden  in  hue;  B.  pyocyaneus,  of  a  greenish-blue;  and 
B.  prodigiosus  which  appears  a  brilliant  red.  7^ 
^  Zymogens  are  the  bacteria  of  fermentation,  which  is  the  chemical 
transformation  of  carbohydrates  by  the  action  of  bacteria,  with  the 
evolution  of  CO 2  CO  &  H.  Such  bacteria  are  useful  in  the  in- 
dustries for  the  production  of  alcoholic  beverages,  wine,  beer,  etc. 
Through  the  actions  of  these  organisms  grape  sugar  is  converted 
into  alcohol,  lactic  acid,  and  acetic  acid.  '^ 

C6H,P,=  2C2H«0+2C02 

glucose        2  alcohol     2  carbonic  acid 


or 


or 


2  lactic  acid 

CqHi206=3  C2H4O2 
2  acetic  acid 
21 


22  PRODUCTS    OF   BACTERIAL   ENERGY 

>v  From  the  bodies  of  ground  yeast  cells  a  soluble  ferment,  Zymase, 
has  been  expressed,  which  causes  alcoholic  fermentation  of  cane, 
and  grape  sugars.  This  fact  proves  that  fermentation  is  not  neces- 
sarily a  vital  process.  VThe  fermentations  of  bacterial  enzymes 
may  give  acids,  and  also  aldehydes,  ketones,  CO 2,  CO,  H,  N,  NH3, 
marsh  gas  and  H2S.  The  carbohydrate  splitting  powers  are  used 
in  determinative  bacteriology.  y<r^-<iXi^  V^viJX^-N^**' ' *^  u:i Aw cj  / 
")k  Fermentation  and  putrefaction  are  bacterIar"eiT23rmic  processes  of 
indispensable  importance  to  life.  Bacteria  reduce  excrementitious 
matters  to  their  elements  and  then  others  build  up  these  elements 
into  conditions  favorable  for  plants.  This  process  affects  the 
cycle  of  utility  of  carbon,  sulphur  and  particularly  nitrogen  in  the 
air  and  soil.  Some  soil  bacteria  can  fix  nitrogen  from  the  air  for 
the  use  of  plants.  Because  of  the  importance  of  these  processes, 
cultures  of  appropriate  bacteria  may  be  spread  upon  exhausted  soil. 
These  are  chiefly  nitrifying  bacteria.  Manure  contains  the  denitri- 
fying organisms.  Bacterial  fermentations  produce  the  flavor  of 
tobacco,  opium  and  butter.. 

sC  Enzyme  Production  by  Bacteria. — ^Ferments  of  great  variety 
and  power  are  formed  by  the  zymogens,  as  proteolytic,  which  dissolve 
proteids,  such  as  casein;  tryptic,  gelatine  liquefying;  diastase,  which 

•  converts  starch  into  s\igaiT)s;invertase,  which  changes  cane  sugar  into 
grape  sugar;  ferments  that  curdle  the  casein  of  milk;  and  it  may  well 
be  tha^Rte  activity  of  pathogenic  bacteria  in  the  body  is  due  to 
ferments  of  some  kind.  The  hemolytic  action  of  the  golden  staphy- 
lococcus or  the  tetanus  bacillus  is  thought,  by  some,  to  be  of  enzymic 
nature.  ^  <kiZ}f\/T>^  ^^  tWcK 

^Organized  ferments  (bacteria/  yeasts)  differ  from  the  unorganized 
(pepsin,  diastase) .  The  latter  "  exercises  solely  a  hydrolytic  action  " 
(Fischer),  causing  the  molecules  of  insoluble  compounds  to  take  up 
water  and  to  separate  into  less  complex  molecules  of  a  different  con- 
stitution, which  are  soluble  in  water.  The  organized  ones  act  differ- 
ently. Highly  complex  molecules  are  split  up,  and  numerous  sub- 
stances of  a  totally  different  character  are  formed  with  the  evolu- 


SAPROGENS  AND  PATHOGENS  23 

tion  of  gases  and  by-products.  (Fischer.)  The  reason  for  this  is, 
perhaps,  to  be  found  in  the  supposition  that  the  bacteria  abstract 
oxygen  for  their  own  use,  and  thus  cause  the  atoms  to  unite  into  an 
entirely  different  substance.y^  According  to  the  above  named  inves- 
tigator, it  is  not  possible  to  express  such  chemical  changes  by  a  sim- 
ple equation.  lExperiments  have  shown  that  B.  typhosus  and  pyo- 
cyaneus  are  able  to  split  up  olive  oil  or  fat,  and  produce  glycerine 
and  fatty  acids,  thus  making  them  accessible  to  fermentation 
(FischerjT]  The  action  of  the  buttermilk  organisms,  while  usually 
very  complex,  may  be  represented  by  the  following: 

Ci2H220ii  +  H20  =  C6Hi20g+C6Hi20g 

lactose  galactose  dextrose 

galactose  lactic  acid 

i^^aprogens  produce  putrefaction  which  is  the  chemical  trans- 
formation of  albuminous  bodies  with  the  evolution  of  nitrogen,  and 
of  alkaloidal  substances,  known  as  ptomainey  Aromatic  elements 
are  also  produced,  such  asindol,  phenol,  kresol,  etc. 

Pathogens. T^If  the  tissues  are  receptive  to  bacteria,  and  if  the 
latter,  in  any  way,  injure  the  tissues,  then  the  invading  organism  is 
called  pathogenies  (fflieoretically  the  tissues  of  the  body  are  sterile. 
But  as  a  matter  of  fact,  isolated  pathogenic  bacteria  such  as  colon 
and  diphtheria  bacilli,  streptococci,  and  pneumococci,  are  often 
found  in  the  tissues  and  cavities  of  the  body,  and  yet  they  cause 
no  pathogenic  changes  or  symptoms.  The  blood  during  life  is 
sterile  in  health,  y, 

>/  Colon  bacilli  have  been  found  encapsulated  in  the  liver  and 
kidneys  of  nondiseased  cadavers,  shortly  after  death,  which  showed 
that  they  had  been  there  some  time.  Sixteen  hours  after  death  the 
blood  and  tissues  teem  with  bacteria  that  have  wandered  in  from 
the  intestines.  It  has  been  shown  that  bacteria,  even  non-motile 
ones,  can  migrate  through  the  body  during  the  agonal  period,  yc 
^Bacteria  may  cause  disease  in  several  ways,  mechanically:  a 


24  PRODUCTS   OF   BACTERIAL   ENERGY 

clump  of  bacteria  may  plug  a  capillary;  or  simply  overwhelm  the 
tissues  and  absorb  the  oxygen  (anthrax) ;  they  may  cause  new  growths 
(tubercle) ;  or  false  membranes  to  form  in  the  larynx  causing  suffo- 
cation (diphtheria)  ;i(ulceration  of  heart  valves  causing  cardiac 
insufficiency;  thrombosis  in  the  veins  and  arteries;  pus  formation;  or, 
by  generating  toxins  that  cause  anaemias,  or  degeneration  of  im- 
portant elements  of  the  nervous  system,  parenchymatous  organs  and 
the  walls  of  the  blood  vessels. 

J  The  tissues  of  certain  animals  are  receptive  for  particular  bacteria, 
and  the  latter  are  therefore  pathogenic  to  that  animal.  B.  of  swine 
plague  is  pathogenic  to  swine,  but  not  to  man.  B,  typhosus  is  patho- 
genic for  man,  but  not  to  swine,  y 
J  As  emphasizd  above,  the  activities  of  bacteria  are  due  to  the 
enzymes  they  produce.  In  the  course  of  their  life,  bodies,  called 
toxins,  are  formed  that  have  the  power  of  producing  illness  in 
higher  plants  and  animals.  These  bodies  are  similar  to  the 
enzymes.  Both  are  produced  in  minute  quantities.  Their  exact 
chemistry  is  not  known,  and  pure  toxins,  at  least,  have  probably 
never  been  isolated.  We  test  for  them  by  animal  experiments 
while  the  presence  of  enzymes  may  be  observed  upon  artificial 
culture  media.  Toxins  of  bacteria  are  not  the  only  ones  formed. 
Castor  bean  produces  a  body  classed  among  the  toxins  as  does 
the  rattlesnake  in  its  venom.  These  bodies  differ  from  ptomaines, 
also  poisons,  by  being  less  resistant  to  heat,  causing  a  peculiar 
blood  reaction  and  by  refusing  isolation  both  of  which  ptomaines 
do  not.  The  toxins  are  not  essential  to  the  life  of  pathogenic 
bacteria  and  some  of  the  usually  virulent  organisms  may  grow 
without  toxin  development.  Toxin  productions  may  be  lost  and 
regained.  The  real  object  of  the  toxins  is  not  known,  as  it  is 
not  thought  that  bacteria  gain  anything  by  producing  disease. 
They  are  separate  from  the  other  chemical  bacterial  products. 
Toxins  may  be  divided  into  those  which  are  secreted  through  the 
bacterial  cell  wall  and  diffuse  through  the  medium  in  which  organ- 
isms are  growing,  the  extracellular  or  soluble  toxins,  and  those  which 


TOXINS  25 

remain  within  the  bacterial  cells  and  are  only  liberated  upon  their 
death  and  disintegration,  the  endotoxins.  Closely  related  to  the 
second  class  are  the  so-called  toxic  bacterial  proteins  or  plasmins. 
These  do  not  separate  from  the  structures  since  bacteria  which 
produce  them  furnish  a  toxic  mass  if  thoroughly  washed,  ground 
and  rewashed.  ^ 

Examples  and  Characters.  [Soluble  or  Extracellular  Toxins. — The 
best  examples  are  those  of  the  tetanus  and  diphtheria  bacillus.  In 
diseases  caused  by  these  germs,  bacteria  do  not  enter  the  body  fluids 
but  the  general  manifestations  are  due  to  absorbed  soluble  poisons. 
Such  toxins  are  soluble  in  water;  they  are  rendered  inert  by  heating, 
sunlight  and  some  chemicals.  They  dialyze  very  slowly  and  are  not 
crystallizable.  They  may  be  precipitated  with  the  albumen  frac- 
tion of  the  medium.  They  may  be  precipitated  and  dried  in  which 
state  they  keep  much  longer  than  when  in  solution,  and  then  are 
more  resistant  to  heat.  Curiously  enough  the  toxins  may  be  de- 
stroyed by  proteolytic  enzymes]  Some  toxins  are  complex;  the 
tetanus  toxin  for  example,  contains  two  elements,  one  a  dissolving 
power  on  red  blood  cells,  the  other  a  stimulator  of  the  motor 
system.  2)  o<.ijjJl*^  / 

l^ndotoxins. — These  are  exemplified  by  the  poisons  of  the  typnoid 
and  plague  organisms.  We  know  little  of  their  chemistry  but  we 
may  assume  that  it  is  of  protein  material  and  similar  to  that  of  the 
bacterial  cell.  These  toxins  are  less  rigidly  specific  than  the  extra- 
cellular poisons.  They  are  probably  quite  complex  in  activity  as 
they  give  rise  to  various  anti-poisons  when  in  the  animal  body. 
These  poisons  are  resistant  to  heating  at  80°  C.  and  keep  under 
artificial  conditions  much  longer  than  soluble  toxinsj 
j[j[he  toxic  bacterial  proteins  are  best  exemplified  by  tuberculin. 
This  is  a  complex  mixture  of  the  proximal  principles  of  the  tubercle 
bacill  us  and  is  probably  albuminose  in  character.  These  substances 
are  almost  as  specific  for  their  own  germs  as  the  toxins  and  much 
more  so  than  the  endotoxins.  They  are  capable  of  producing  a 
reaction  in  animals  similar  to  that  which  might  be  produced  by  the 


26  PRODUCTS   OF   BACTERIAL   ENERGY 

organisms  themselves.  For  example  tuberculin,  wholly  free  from 
tubercle  bacilli,  will  produce  a  reddening  of  the  skin  or  a  rise  of 
temperature  if  injected  into  a  tuberculous  individual.  The  reac- 
tions from  mallein  and  luetin  {q.v.)  injection  are  due  to  toxic  pro- 
teiBSfl  They  are  thermostable,  that  is  not  destroyed  at  ioo°  C. 
TK^is  also  called  coctostabile. 

In  practice  it  may  not  be  so  simple  to  separate  bacteria  that 
produce  the  various  poisonous  elements  as  the  above  descriptions 
would  indicate.  Toxins  are  all  in  a  sense  specific.  That  is  they 
are  for  the  most  part  selective  in  action.  The  diphtheria  toxin  is 
absorbed  from  a  raw  inflamed  surface  under  cover  of  an  exudate 
composed  of  fibrin  and  bacteria.  The  tetanus  toxin  is  absorbed 
from  its  seat  of  manufacture  in  the  depths  of  a  punctured  wound. 
They  are  harmless  if  swallowed.  The  endotoxin  of  typhoid  bacilli 
has  no  pathogenic  effect  if  swallowed  or  rubbed  in  skin  or  mucous 
membrane.  If  it  be  injected  under  the  skin  in  the  absence  of 
bacteria  it  will  call  forth  reactions  on  the  part  of  the  body  similar 
to  those  expressed  when  living  typhoid  germs  are  circulating. 
Toxins  are  again  relative  in  their  affinities.  Tetanus  toxin  is  fatal 
for  man  and  horses  while  rats  and  birds  are  resistant  to  it.  We 
use  this  expression  of  specificity  for  determiding  the  nature  of 
certain  germs.  We  may  speak  of  these  failures  to  react  as  fail- 
ures of  receptivity  on  the  part  both  of  the  microbe  and  the  injected 
animal. 


CHAPTER  III. 

INFECTION. 

7  Infection  means  the  successful  invasion  of  the  tissues  of  the  body 
by  either  animal  (protozoa,  vermes)  or  vegetable  (bacteria  and 
moulds)  organisms  with  the  evidences  of  their  action.  To  success- 
fully infect  the  body,  bacteria  must  enter  the  tissues,  be  of  sufficient 
number,  find  the  tissues  receptive,  and  continue  to  multiply. 

The  skin,  mucous  membranes,  and  the  various  cavities  of  the  body 
connected  with  the  outside  air,  teem  with  countless  bacteria  at  all 
times,  many  of  which  are  pathogenic,  yet  there  is  no  infection,  be- 
cause the  tissues  are  not  invaded.  Again,  there  can  be  no  doubt 
that  highly  pathogenic  bacteria  enter  the  tissues  of  healthy  people 
at  times,  in  small  numbers,  and  yet  no  disease  is  produced,  because 
of  their  scarcity,  or  by  reason  of  the  tissues  not  being  receptive. 
Infection  implies  not  only  invasion  of  the  body,  but  injury  to  the 
tissue.  Certain  bacteria  may  invade  a  body,  and  yet  create  no 
harm.  These  bacteria  may  enter  dead  or  dying  body  tissues,  and 
secrete  poisonous  substances  (toxins)  which  may  be  absorbed,  and 
produce  pathologic  symptoms  known  as  Saprcemia.  Clots  of 
blood  in  the  parturient  uterus,  and  gangrenous  limbs  may  be  in- 
vaded by  strict  saprophytes  incapable  of  life  in  living  tissues,  and 
yet  cause  much  harm  by  the  absorption  of  their  products. 

Infestation  is  when  bacteria,  even  pathogenic,  are  present  in  a 
place  without  exciting  a  reaction.  Matter  carrying  pathogenic 
germs  is  called  infective. 

Depending  upon  their  ability  to  grow  in  the  body,  bacteria  may 
be  divided  into  (i)  purely  saprophitic;  (2)  occasionally  parasitic; 
and  (3)  purely  parasitic.     A  host  harbors  a  parasite. 

Purely  saprophitic  germs  cannot  live  in  tissues  at  all;  those  that 

27 


28 


INFECTION 


are  occasionally  parasitic  lead  a  saprophitic  existence  in  the  soil 
or  water,  and  yet  may  invade  the  body,  and  produce  disease:  the 
tetanus  and  malignant  oedema  bacilli  are  examples  of  this  group. 
Those  bacteria  that  are  purely  parasitic  are  only  known  as  they  exist 
in  the  tissues  of  the  infected  host,  and  have  no  outside  existence 
at  all. 


Kruse's  Scheme  Illustrating  the  Action  of  Various  Parasitic 

Bacteria. 


Parasitic 
Bacteria. 


A 

Occasionally  paras- 
itic. Such  as  the  Tetan- 
us bacillus. 


B 

Always  parasitic  only 
found  in  the  lesions  of 
disease.  Such  as  the 
B.  tuberculosis. 


1.  Local  infection  due  to  the 
ability  of  the  organism  to 
take  on  unrestricted  growth. 

a.  Surface  inflammation ^ 
boils;  staphylococci. 

b.  Surface  inflammation 
with  extension  of  con- 
tinuity; erysipelas 
streptococci. 

c  Surface  infection  with 
marked  toxin  products; 
diphtheria. 

d.  Deep  focal  inflamma- 
tion; tubercles. 

2.  General  infection  of  un- 
restricted growth. 

a.  By  continuous  infec- 
tion; glanders. 

b.  Metastases,  as  in  py- 
comia. 

c.  By  universal  rapid 
growth  and  invasion, 
as  in  sepsis  and  an- 
thrax. 


koch's  postulates  29 

Koch*s  Postulates. 

In  order  to  prove  that  a  certain  organism  is  the  infectious  agent 
of  a  given  disease,  Koch  has  devised  four  postulates  which  the  given 
organism  must  fulfill  before  it  can  be  considered  the  cause  of  the 
disease. 

1.  The  organism  must  be  found  microscopically  in  the  tissues 
of  the  animal  having  the  disease,  and  its  position  in  the  lesion  should 
explain  the  latter. 

2.  It  must  be  isolated  in  pure  state  from  bodies  of  the  diseased 
animals. 

3.  And  then  it  must  be  grown  for  successive  generations  in  cul- 
ture media. 

4.  If  injected  into  a  healthy  animal,  or  animals,  it  must  produce 
the  same  disease,  and  be  found  in  the  lesions  of  the  disease  in  the 
animal's  tissues,  y/ 

Some  of  the  many  organisms  that  certainly  fulfil  these  conditions, 
are  as  follows: 


Streptococcus  Pyogenes  (Sepsis).    Actinomyces. 

B.  of  Tuberculosis.  B.  of  Diphtheria. 

B.  of  Anthrax.  B.  of  Tetanus. 

B.  of  Glanders.  B.  of  Malignant  Oedema. 

B.  of  Bubonic  Plague.  B.  of  Malta  Fever. 

B.  of  Typhoid.  B.  of  Dysentery. 

Spirillum  cholerce.  Meningococcus. 

Pneumococcus  {Pneumonia).         B.  of  Leprosy. 
SpirochcBta  of  Relapsing  Fever  and  of  Syphilis. 
There  are  several  other  organisms  that  are  considered  to  be  the 
cause  of  specific  disease,   but  they  do  not  fulfil  the  postulates. 
Among  these  are: 

The  Organism  of  Scarlet  Fever  (Protozoans). 

The  Protozoa  of  Malarial  Fever. 

Amoeba  Dysenterice. 


3©  INFECTION 

In  rheumatic  fever,  measles,  whooping-cough,  poliomyelitis, 
mumps,  yellow  fever,  typhus  fever,  chicken-pox,  rabies,  and  dengue, 
the  specific  cause  has,  thus  far,  eluded  discovery.  In  the  case  of 
yellow  fever  (Reed  and  Carrol)  and  hog  cholera,  it  has  been  found 
that  the  cause  of  these  diseases  resides  in  the  blood,  and  if  the 
serum  of  the  latter  is  carefully  filtered  through  a  Berkefeld  filter, 
it  is  still  capable  of  producing  the  disease  in  susceptible  animals. 
Careful  microscopic  search  fails  to  show  any  bodies  in  the  serum 
that  might  be  considered  the  agents  of  infection,  and  it  is  thought 
that  these  organisms  are  submicroscopic. 

If  the  invading  organism  is  a  pure  saprophyte  the  various  forces 
for  internal  defence  immediately  act  upon  and  destroy  it.  If  it  is 
pathogenic  for  other  animals,  their  defensive  agencies  have  no  effect 
upon  it  in  their  tissues,  but  in  the  human  body  the  bacteriolysins 
dissolve  it,  or  the  phagocytes  devour  it  and  carry  it  away.  The 
liver,  according  to  Adami,  destroys  at  once  bacteria  absorbed  from 
the  intestines. 

Bacteria  are  disposed  of  in  divers  ways,  by  means  of  the  lymph 
channels  they  are  carried  to  the  various  mucous  surfaces  of  the 
body,  intestinal  and  bronchial.  During  typhoid  fever,  the  typhoid 
bacilli  are  often  found  in  the  urine.  The  kidneys  at  least  allow 
the  escape  of  some  organisms  from  the  blood.  Pathogenic  bacteria 
are  discharged  from  the  body  in  feces,  pus,  sputum,  and  in  scales 
in  the  desquamating  skin  diseases. 

To  successfully  inoculate  a  guinea  pig  with  tuberculosis,  the 
tubercle  bacilli  should  be  injected  beneath  the  skin. 

In  working  with  infections  produced  by  the  B.  proteus  vulgaris, 
it  was  found  by  Watson  Cheyne  that  6,000,000  bacilli  injected 
under  the  skin,  did  not  produce  any  lesion;  8,000,000  formed  an 
abscess;  56,000,000  gave  rise  to  a  phlegmon;  and  225,000,000  were 
necessary  to  cause  death  in  two  hours. 

In  experimenting  with  the  staphylococcus  aureus,  it  was  found 
that  250,000,000  were  required  to  cause  an  abscess;  and  1,000,000,- 
000  were  needed  to  cause  death.     The  internal  powers  of  defense 


ATTENUATION  OF  BACTERIA  3 1 

were  able  in  each  case  to  cope  with  or  limit  the  action  of  a  few  mil- 
lion to  a  certain  locality,  but  could  not  withstand  the  injection  of 
overwhelming  numbers,  which  caused  the  animal's  death. 

Bacteria  to  be  successfully  infectious  must  be  virulent.  Viru- 
lence is  best  described  as  the  power  of  a  parasite  to  invade  and  grow 
within  the  body  by  resisting  its  natural  defenses,  and  gradations 
depend  upon  the  ratio  of  these  two  forces  (Wolff  Eisner).  Pfeiffer's 
explanation  of  virulence  assumes  that  bacteria  have  binding  posts 
or  receptors  and  the  more  of  these  a  germ  has,  the  more  of  the 
natural  defenses  it  can  anchor  and  remove  from  the  field.  Their 
virulence  can  be  lessened  by  cultivation  at  a  higher  temperature 
than  the  body,  42.5°  C.  to  47°  C;  by  drying;  the  exposure  to  light; 
the  action  of  chemicals;  compressed  oxygen;  and  by  passing  the  or- 
ganism through  the  bodies  of  non-susceptible  animals.  The  atten- 
uation or  weakening  of  the  pathogenic  powers  of  bacteria  is  useful 
for  the  production  of  various  vaccines  which  are  valuable  in  preven- 
tive medicine. 

By  growing  the  anthrax  bacillus  at  a  high  temperature,  42.5°  C, 
it  becomes  so  avirulent  that  it  is  incapable  of  destroying  sheep  or 
rabbits.  It  is  then  used  as  a  vaccine  to  prevent  infection  with 
virulent  bacilli.  By  exposing  the  spinal  cords  of  animals  dead  from 
hydrophobia  to  the  action  of  drying  for  various  periods,  Pasteur  was 
able  to  attenuate  the  virus,  so  that  it  would  not  produce  hydro- 
phobia, but  on  the  contrary,  it,  by  repeated  inoculation,  caused 
immunity.  The  inoculation  of  monkeys  (which  are  non-suscept- 
ible) with  hydrophobia  virus  attenuates  it.  The  growth  of  the  small- 
pox organism  in  the  cow,  causing  cow-pox,  so  reduces  the  virulence 
of  the  germ  that  it  is  incapable  of  producing  small-pox  in  man,  but 
only  vaccinia;  infection  with  this  gives  immunity  against  small-pox. 
The  flesh  of  animals  that  have  died  from  quarter-evil  is  so  changed 
by  heat  and  desiccation  that  if  it  is  injected  into  susceptible  animals, 
they  do  not  succumb  but  are  vaccinated  against  infection  with  the 
virulent  organism. 

When  we  speak  of  attenuation  of  virulence  we  usually  refer  to  the 


32  INFECTION 

effects  on  experimental  animals  and  specify  what  attenuation  is 
meant  when  they  are  to  be  used  as  vaccine.  A  very  interesting 
virulent,  yet  attenuated,  form  of  streptococcus  is  to  be  met  in  sub- 
acute endocarditis.  These  organisms  produce  serious  or  even  fatal 
valvulitis,  and  yet  have  no  effect  upon  other  organs  or  upon  lower 
animals.  They  are  extremely  hard  to  remove  from  the  body. 
They  have  accustomed  themselves  to  residence  in  the  body,  have 
established  a  balance  or  poise  between  their  offenses  and  the  bodily 
defenses  and  practically  cannot  be  rapidly  dislodged.  These  are 
called  fixed  or  fast  strains.  Such  strains  may  be  seen  under  other 
conditions  such  as  the  typhoid  bacillus  in  the  gall  bladder.  These 
fast  strains  usually  are  found  at  places  remote  from  intimate 
opposition  of  leucocytes  and  blood  serum  as  in  the  cases  cited. 

The  malignancy  of  bacteria  may  be  heightened  in  various  ways: 
(i)  By  passing  them  repeatedly  through  the  bodies  of  susceptible 
animals;  (2)  by  cultivation  in  culture  media  in  collodion  sacs 
placed  in  the  abdominal  cavities  of  animals;  (3)  by  injections  mixed 
with  other  injurious  substances,  such  as  lactic  acid,  and  the  meta- 
bolic products  of  foreign  bacteria.  Cultures  of  pneumococci  may 
be  made  so  virulent  by  the  first  means  that  only  one  pneumococcus 
is  capable  of  setting  up  a  fatal  septicaemia  in  a  rabbit.  By  injecting 
attenuated  diphtheria  bacilli  with  streptococci  into  a  rabbit,  the 
virulence  of  the  bacilli  can  be  raised,  as  mixed  infection  often  adds 
to  the  virulence  of  an  organism.  Malignant  streptococcic  infection 
added  to  virulent  diphtheria  infection,  greatly  increases  the  severity 
of  the  disease. 

The  secondary  streptococcic  infection  in  small-pox  and  in 
phthisis  complicates  the  primary  infection  and  frequently  causes 
death  of  the  individual  affected.  The  hectic  fever  and  sweats  of 
phthisis  are  due  to  this  secondary  infection.  Combinations  of 
diphtheria  bacilli  and  pneumococci  increases  the  virulence  of  the 
latter.  The  transference  of  infection  agents  from  one  person  to 
another  during  an  epidemic  increases  the  virulent  action  of  the 
organism  by  reason  of  the  rapid  passage  from  individual  to  individ- 


AVENUES  OF  INFECTION  33 

ual.  Two  infections  may  occur  simultaneously,  each  preserving 
separate  characteristics,  and  perhaps  aggravating  each  other. 

The  avenue  of  infection  and  the  tissues  infected  alter  the  type  of 
the  disease  exceedingly.  Streptococci  invading  the  tonsils  cause 
tonsillitis,  but  the  same  organisms  entering  the  skin  cause  erysipelas 
or  phlegmons;  or  if  the  uterus  is  infected  after  the  birth  of  a  child 
the  disease  is  stil]  different  and  more  serious.  If  the  tubercle  bacilli 
enter  the  skin  they  produce  lupus;  if  swallowed  they  cause  ulceration 
of  the  bowels,  and  subsequently  invade  the  peritoneum;  if  inhaled, 
tuberculosis  of  the  air  passages,  phthisis,  or  tubercular  laryngitis 
may  follow.  If  cholera  spirilla  be  injected  into  a  vein  of  a  guinea 
pig,  it  may  develop  choleraic  septicaemia;  if  they  are  injected  into 
the  peritoneal  cavity,  a  choleraic  inflammation  of  the  peritoneum  is 
produced,  and  not  a  septicaemia.  Pneumococci  if  injected  into  a 
vein  cause  a  rapid  septicaemia,  or  they  may  give  rise  to  abscesses 
anywhere  in  the  body.  Like  streptococci,  they  may  be  the  cause 
of  inflammation  in  any  tissue,  particularly  serous  membranes,  and 
show  different  clinical  entities,  according  to  the  organs  involved,  and 
the  morbid  anatomy  and  physiology  produced.  The  fatality  of  a 
bacterial  infection  varies  with  the  avenue  of  inoculation:  it  is  safer  to 
have  a  skin  infection  than  a  meningeal,  or  endocardial  one,  not  only 
from  the  likelihood  of  rapid  toxin  absorption,  but  from  purely 
mechanical  damage,  as  pressure  and  interference  with  vital  functions 
by  inflammatory  products  such  as  exudates,  tubercles,  serum  and 
pus.  The  injection  of  pneumococci  under  the  skin  of  a  dog  has  a 
more  rapidly  fatal  effect  than  when  they  are  injected  into  a  vein, 
according  to  Klemperer. 

It  seems  practically  proven  now  that  tubercle  bacilH  may  enter 
the  lung  by  way  of  the  intestinal  tract,  but  Ghon  has  lately  shown 
that  tuberculosis  in  childhood  usually  starts  as  an  infection  directly 
into  the  lung  tissue  by  bacilli  coming  in  with  air. 

Local  immunity  to  infection.  There  is  evidently  more  resistance 
offered  by  the  liver  against  invasion  than  by  the  peritoneum.  It  is 
not  likely  that  a  man  would  contract  typhoid  through  skin  infection, 
3 


34  INFECTION 

nor  is  it  probable  that  he  would  contract  tetanus  by  swallowing 
tetanus  bacilli,  but  the  reverse  of  these  conditions  certainly  produces 
infection. 

Infection  may  be  caused  from  without  the  body,  or  from  within. 
Lockjaw,  sepsis,  hydrophobia,  or  anthrax  may  follow  injuries  from 
rusty  nails,  splinters,  weapons,  unsterile  fingers,  or  instruments. 
Personal  intercourse,  bites,  kisses,  sexual  intercourse,  association 
with  persons  suffering  from  exanthematous  or  contagious  diseases 
may  transmit  disease. 

Winslow  has  found  colon  bacilli  upon  9  percent  of  the  hands  he 
examined.  Tubercle  bacilli  have  been  found  on  the  hands  of  the 
non-tuberculous.  Some  organisms,  notably  the  smegma  bacillus, 
pyocyaneus  bacilli  and  cocci  resembling  the  white  pus  former,  may 
be  said  to  be  normal  inhabitants  of  the  skin. 

The  bites  of  insects  that  are  intermediate  hosts  of  infectious  agents 
(plague  bacilli,  malarial  organisms,  etc.)  are  sources  of  infection 
from  without,  as  is  also  the  ingestion  of  infected  food  or  water. 

Infection  from  within  may  be  caused  by  the  migration  of  bacteria 
from  the  skin  inwards,  or  from  any  of  the  mucous  membranes,  on 
which,  and  in  which  many  pathogenic  bacteria  at  all  times  may  be 
found. 

Bacteria  from  the  mouth,  stomach,  intestines  and  the  rectum  may 
invade  the  tissues  and  the  blood  under  certain  conditions.  This  is 
particularly  the  case  during  the  last  stages  of  diseases,  not  necessarily 
infectious,  such  as  chronic  heart  disease,  kidney  disease,  or  diabetes. 
Vital  resistance  is  much  lowered,  and  intestinal  bacteria,  invading 
the  tissues  in  enormous  numbers,  set  up  what  is  known  as  terminal 
infection,  which  is  often  the  immediate  cause  of  death. 

The  stomach  with  its  gastric  juice,  containing  during  digestion 
.2  percent  to  .3  percent  of  hydrochloric  acid,  guards  the  lower  ali- 
mentary tract  against  infection.  A  great  many  bacteria  are  ingested 
with  foods,  particularly  with  milk,  cheese,  and  over-ripe  fruit. 
These  in  the  most  part  are  quickly  destroyed  by  the  hydrochloric 
acid.     When  the  stomach  is  diseased  and  the  contents  become 


AVENUES  OF  INFECTION  35 

stagnant,  as  in  stenosis  of  the  pylorus,  and  in  carcinoma,  when  HCl 
is  diminished,  or  absent,  fermentative  bacteria  give  rise  to  great 
amount  of  gas,  and  lactic  acid,  to  the  great  discomfort  of  the  patient. 
The  normal  acidity  of  the  stomach  is  a  great  safe-guard  against 
infection  with  cholera.  If  tubercle  bacilli  are  swallowed,  and  if 
infection  occurs,  the  lesion  is  not  always  localized  to  the  alimentary 
tract.  Lesions  of  the  lymph  glands,  peritoneum,  bones,  and 
nervous  tissues  often  follow  the  ingestion  of  these  organisms.  Dogs 
fed  on  soup  containing  great  numbers  of  tubercle  bacilli,  and  then 
killed  three  hours  after,  were  found  to  have  bacilli  in  the  thoracic 
duct.  Chyle  from  the  duct,  injected  into  guinea  pigs,  caused 
tuberculosis  in  them  (Nicolas  and  Descos).  Cholera  and  typhoid 
organisms  thrive  in  intestinal  contents,  elaborating  poisons  which 
greatly  depress  the  individual. 

The  interior  of  the  uterus,  the  bladder,  urine,  and  deep  urethra, 
are  generally  sterile  in  health.  With  the  exceptions  noted  where 
germs  are  not  usually  found,  all  tissues,  especially  the  inlets  and 
outlets  of  the  body,  may  be  said  to  have  a  normal  bacterial  flora. 

The  placenta  is  an  avenue  of  infection  in  several  diseases:  notably 
small-pox,  anthrax,  glanders,  typhoid  fever,  and  sometimes  tuber- 
culosis pass  through  the  placenta  from  mother  to  foetus.  Strep- 
tococci may  pass  through  the  placenta  of  a  woman  with  ante-deliv- 
ery sepsis  and  cause  peritonitis  in  the  child.  Recurrent  fever  has 
been  transmitted  from  mother  to  foetus,  and  the  specific  spirillum 
has  been  detected  in  the  latter's  blood. 

A  case  has  been  recorded  in  which  a  woman  suffering  from  pneu- 
monia gave  birth  to  a  child,  which  died  thirty-six  hours  afterward, 
and  autopsy  revealed  a  consolidation  of  the  lower  left  lung,  and 
microscopic  examination  discovered  pneumococci.  A  hydrophobic 
cow  was  delivered  of  a  calf  that  developed  rabies  three  days  after 
birth. 

McFarland  divides  microbic  infection  in  three  heads: 

Phlogistic.  Characterized  by  restricted  growth  and  local  irrita- 
tion. 


36  INFECTION 

Toxic.  Characterized  by  restricted  growth  and  toxin  dissemi- 
nation. 

Septic,  Characterized  by  unrestricted  growth  in  the  blood  and 
lymph.  In  the  three  groups,  the  damage  is  done  ultimately,  by 
metabolic  products  acting  on  the  tissues.  If  the  product  is  not 
soluble  the  harm  done  is  purely  local,  as  in  the  formation  of  tubercles 
by  the  toxin  of  the  tubercle  bacilli. 

If  the  growth  is  restricted,  as  in  tetanus  and  diphtheria,  the  toxin 
being  soluble  and  diffusible,  harm  is  done  to  tissues  remote  from 
the  infected  area. 

Anthrax  and  streptococci  and  other  pus  organisms  by  rapid 
increase  in  the  blood  eventually  infect  all  the  tissues. 

Combinations  of  these  forms  of  infection  may  be  at  first  confined 
to  some  particular  area  like  the  pneumococcus,  which  are  generally 
restricted  to  the  lungs  at  the  outset,  but  ultimately  they  infect  the 
blood,  causing  septicaemia  and  localized  lesions  in  more  or  less 
remote  parts,  such  as  the  veins  of  the  leg,  or  inflammation  of  the 
meninges. 

Soluble  products  of  bacterial  activity  which  are  alkaloidal  (basic), 
crystalline  in  character,  and  mostly  poisonous,  are  known  as  pto- 
maines, or  putrefaction  alkaloids.  They  are  highly  complex  in 
chemical  structure,  and  are  difficult  to  isolate. 

Certain  albuminoid  bodies,  products  of  bacterial  activity,  known 
as  toxins,  are  produced  by  several  pathogenic  bacteria. 

Those  that  are  essentially  bound  up  in  the  protoplasm  of  the 
bacteria  itself,  are  known  as  intracellular  toxins,  and  bacteria  plas- 
mins.  The  tubercle  bacillus,  and  other  members  of  the  acid-fast 
group,  the  colon  and  typhoid  bacillus,  and  the  cholera  spirillum  all 
contain  these.  They  may  be  extracted  by  freezing  the  organism 
with  liquid  air,  and  grinding  it  while  frozen  and  brittle,  or  by  simply 
grinding  it  with  sterile  sand  and  water.  The  new  T.R.  tuberculin 
belongs  to  this  group  of  toxins. 

Bacterioprotein  or  plasmins  are  albuminous  bodies  produced 
by  bacteria  that  are  not  altered  by  heat,  and  which  produce  fever 


TOXINS  OR  TOXALBUMINS  37 

and  inflammation.  The  best  examples  of  these  are  mallein,  a 
product  obtained  from  old  cultures  of  glanders  bacilli,  and  the 
original,  or  old  tuberculin  of  Koch. 

Toxins  or  toxalbumins  are  soluble  bacterial  products  which  are 
removable  by  filtration  from  the  bacteria,  and  which  are  thermola- 
bile.     The  tetanus  and  diphtheria  toxins  belong  to  this  class. 

These  various  poisons  produce  many  of  the  clinical  pathological 
entities  and  symptoms,  known  to  physicians.  Their  highly  com- 
plex molecular  structure  enables  a  group  of  atoms  in  the  toxic  mole- 
cule to  unite  with  a  certain  other  group  of  atoms  in  the  protoplasmic 
molecule  of  a  body  cell.  The  latter  is  either  killed  outright,  or  else 
is  stimulated  to  produce  other  free  groups  of  combining  atoms  (lat- 
eral chains)  which  may  unite  with  other  toxic  groups. 

Various  kinds  of  cells  are  attacked  in  infective  processes.  Leuco- 
cytes may  be  degenerated,  forming  pus;  red  blood  cells  may  be  dis- 
solved, causing  anaemia;  important  nerves  may  be  degenerated;  or 
muscle  fibers  of  the  heart  may  undergo  fatty  degeneration  and  die. 
Again,  mechanically  important  serous  cavities  may  be  filled  with 
serum,  interfering  with  normal  functions  of  the  enveloped  organs. 
The  heart  orifices  may  be  closed  partially  or  emboli  may  form,  or 
false  membranes  block  the  air  passages,  and  a  hundred  other  patho- 
logical changes  may  be  wrought  by  these  toxins. 

If  toxins  are  injected  into  the  body  with  the  specific  organism  pro- 
ducing them,  the  effect  of  the  latter  is  greatly  increased.  Tetanus 
spores,  washed  free  of  toxins,  if  injected,  are  incapable  of  setting  up 
tetanus. 

Most  toxins  are  easily  decomposed  by  sunlight,  air,  and  heat. 
Absolute  alcohol  separates  the  active  principle  from  the  bouillon 
in  which  it  grows.  Ammonium  sulphate  also  separates  the  toxins 
of  tetanus  and  diphtheria  bacilli,  which  float  on  top  of  the  fluid,  from 
which  they  may  be  collected,  dried  and  powdered,  and  in  this  state' 
may  be  kept  much  longer  without  deteriorating  into  inert  substances. 
Small  quantities  of  bile  and  pancreatic  juice  destroy  the  toxic  proper- 
ties of  diphtheria  and  tetanus  toxin. 


38  INFECTION 

If  toxin  and  anti-toxin  (see  immunity  chapter)  are  mixed  in  rela- 
tive proportions,  chemical  neutralization  takes  place.  Since  the 
toxins  cannot  be  isolated  in  a  chemically  pure  form,  their  exact 
composition  cannot  be  known,  except  by  studying  their  effects  upon 
animals  and  animal  tissues.  Hence,  when  anti-toxin,  added  to 
toxin  in  a  test-tube  is  injected  into  an  animal,  and  no  harm  results, 
it  is  rightly  assumed  that  the  toxin  is  neutralized,  and  both  are 
chemically  bound;  yet  if  fresh  toxin  is  added  to  the  mixture,  it  is  no 
longer  neutral. 

If  the  toxin  of  the  pyocyaneus  and  the  anti-toxin  be  mixed  so 
that  they  neutralize  each  other,  and  if  the  mixture  is  heated,  the 
neutralization  disappears,  and  the  mixture  becomes  toxic  again. 
That  the  union  is  a  chemical  one,  may  be  inferred  from  the  fact 
that  it  is  more  rapid  in  concentrated  solution  than  in  weak,  and  is 
much  quicker  when  warmed  than  when  cold,  and  it  follows  the  law 
of  multiples,  one  part  toxin  neutralizing  one  part  of  anti-toxin,  and 
ten  parts  of  toxin  neutralizing  ten  parts  of  anti-toxin.  All  this  is  in 
accord  with  chemical  laws.  Toxins  sometimes  degenerate  into 
what  Ehrlich  has  called  toxoids,  substances  that  bind  (unite  with) 
anti-toxin  just  as  effectively  as  toxins,  while  they  are  not  poisonous, 
yet  may  stimulate  healthy  cells  to  secrete  anti-toxins  if  they  are 
injected  into  the  body  of  experiment  animals. 

More  is  known  about  the  toxins  of  diphtheria  and  tetanus  bacilli 
than  of  any  other.  Diphtheria  toxin  has  numerous  component  sub- 
stances, one  of  which  is  the  toxin  that  causes  the  acute  phenomena 
of  diphtheria  intoxication.  Another,  toxon,  causes  cachexia  and 
paralysis  some  time  after  infection. 

Tetanus  toxin  is  composed  of  two  substances;  tetanospasmin  and 
tetanolysin.  The  first  unites  chemically  with  the  motor  elements 
of  the  nervous  system,  producing  degeneration  and  causing  tremen- 
dous contractions  of  the  muscles  governed  by  the  nerves  involved. 
The  second  has  the  property  of  dissolving  tissues,  such  as  blood  cells. 

Tetanus  toxin  travels  from  the  infected  site  to  the  cord  by  way  of 
the  nerves;  it  is  exceedingly  poisonous;  a  single  prick  of  the  finger 


AGGRESSINS  39 

with  a  needle  moistened  with  toxin,  has  induced  tetanic  symptoms 
If  tetanus  toxin  of  known  strength  is  mixed  in  a  test-tube  with 
fresh  brain  substance  of  a  guinea  pig,  the  toxin  is  no  longer  toxic 
for  guinea  pigs.  This  shows  that  there  is  a  chemical  union  of  the 
toxin  and  the  cells  of  the  brain.  Cells  of  other  organs  have  no  such 
effect.     This  explains  specific  action  of  tetanus  upon  nervous  tissue. 

Aggressins. 

If  tubercle  bacilli  are  injected  into  the  abdominal  cavity  of  a 
guinea  pig,  rapidly  fatal  tuberculosis  is  produced.  If  the  exudate 
produced  in  the  peritoneum,  consisting  of  lymphocytes,  is  sterilized 
and  injected  into  another  guinea  pig,  together  with  some  virulent 
tubercle  bacilli,  the  animal  will  succumb  in  twenty-four  hours.  If 
the  exudate  alone  is  injected  no  effect  will  follow;  if  bacilli  alone  are 
injected,  a  tuberculous  peritonitis  will  be  produce  in  a  few  weeks. 
It  is  the  exudate  plus  bacilli  that  does  the  harm.  The  exudate  is, 
in  this  instance,  the  aggressin.  Bail,  who  originated  the  doctrine  of 
aggressins,  believes  that  a  bacteriolysin  is  produced,  which,  acting 
on  the  bacilli,  liberates  an  endotoxin,  which  paralyzes  the  poly- 
nuclear  leucocytes,  inhibiting  their  action  as  phagocytes. 

By  heating  the  exudate  to  60°  C.  the  aggressins  are  increased  in 
activity,  and  it  has  been  found  that  small  amounts  are  relatively 
stronger  than  larger  ones. 

This  phenomenon  has  been  explained  by  Bail  in  this  way.  He 
assumes  that  there  are  two  substances  in  the  exudate,  one  is  thermo- 
labile,  which  prevents  rapid  death,  the  other  is  thermostabile  and 
this  is  favorable  to  rapid  death. 

Bail  assumes  that  a  tubercular  cavity  in  an  animal  contains  a 
great  amount  of  the  aggressin,  which  prevents  chemotaxis  of  the 
ploynuclear  leucocytes,  but  not  of  the  mononuclears  or  lymphocytes. 

In  the  peritoneal  cavity  without  aggressins,  into  which  tubercle 
bacilli  have  been  injected,  an  active  phagocytosis  at  once  is  begun 
by  the  polynuclears,  and  the  injected  bacilli  are  in  a  great  measure 


40  INFECTION 

destroyed,  and  those  left  develop  more  slowly,  producing  a  tuber- 
culosis in  normal  course  of  time.  It  is  possible  to  immunize  animals 
against  this  aggressin  producing  an  anti-aggressin,  which  substance 
will  not  only  neutralize  the  aggressin  but  also  stimulate  the  leuco- 
cytes to  phagocytosis. 

This  aggressin  theory  has  been  apphed  to  other  infections  with 
like  results,  notably  in  pneumococcus,  typhoid,  dysentery,  and 
plague  infection. 


CHAPTER  IV. 


/ 


IMMUNITY. 


By  immunity,  is  understood  the  inherent  power  of  a  living  body 
to  successfully  withstand  the  invasion  of  infective  agents,  e.g.,  bac- 
teria, or  such  deleterious  and  toxic  substances  as  toxins,  drugs,  com- 
plex poisonous  albumins,  snake  venom,  foreign  blood  sera,  etc.  f^ 

The  following  tables  will,  perhaps,  be  helpful  in  the  study  of  the 
subject. 


v.. 


V'<^AA. 


I.  Immunity 


2.  Immunity 


Natural 
Acquired 


Racial  immunity 
Inherited  immunil 
Active  immunity 
Passive  immunity 


Anti-toxic  ^t;A^.^o^ 

Anti-bacterial    *^<^A^IU!j^,r^  &    I 


It  is  a  well  known  fact  that  one  attack  of  an  infectious  disease 
generally  protects  an  individual  against  a  subsequent  attack.  It 
has  also  been  known  for  centuries  that  the  human  system,  by  first 
taking  very  small  doses,  and  gradually  increasing  them,  can  be  so 
accustomed  to  poison,  that  large,  and  otherwise  deadly  quantities 
may  be  taken  at  one  time  with  impunity.  Among  the  poisonous 
substances  to  which  men  can  accustom  themselves  are:  tobacco, 
morphia,  arsenic,  and  alcohol.  Animals  treated  in  a  like  manner 
also  become  immunized  to  powerful  toxins,  snake  venom,  etc. 

Natural  Immunity. — The  hog  is  immune  to  snake  venom;  the 
chicken  to  tetanus.  Man  is  immune  to  hog,  or  chicken  cholera. 
The  negro  is  not  so  susceptible  to  yellow  fever  as  is  the  white.  Ani- 
mals cannot  be  infected  with  scarlet  fever,  malaria,  and  measles. 

41 


42  IMMUNITY 

Young  adults  are  more  susceptible  to  typhoid  fever  than  are  elderly 
ones.  Infants  are  exceedingly  prone  to  suffer  from  milk  infection 
while  older  children  are  not.  Certain  diseases  are  known  as  chil- 
dren's diseases,  because  adults  rarely  have  them.  Again,  one 
individual  may  contract  a  disease,  while  another  exposed  at  the 
same  time  will  not. 

Acquired  Immunity. — Actively  acquired  by  infection.  One 
attack  of  yellow  fever  immunizes  the  individual  against  subsequent 
attacks.     Vaccination  actively  immunizes  against  small-pox. 

Passively  acquired.  Actually  injecting  protective  substances 
(anti-toxic  sera)  into  the  blood.  The  immunity  against  a  given 
disease  (diphtheria)  resides  in  the  anti-toxic  sera. 

Immunity  is  nearly  always  relative.  A  small  quantity  of  toxin 
may  be  innocuous,  while  a  large  quantity  may  cause  a  fatal  toxaemia. 

There  have  been  several  theories  advanced  to  account  for  the 
various  phenomena  of  immunity. 

Exhaustion  Theory. — Pasteur  conceived  that  bacteria  as  they 
grow  in  the  body,  use  up  or  exhaust  something  vitally  necessary  to 
the  subsequent  growth  of  that  particular  kind  of  bacteria. 

Retention  Theory. — It  was  held  that  some  noxious  agent  was 
retained  by  the  body  which  prevented  the  further  growth  of  bacteria. 

The  modern  conception  of  immunity  deals  with  two  theories, 
the  theory  of  phagocytosis  of  Metchnikoff,  which  may  be  termed 
the  cellular  or  biologic  one,  and  the  lateral-chain,  or  the  humoral 
or  chemical  theory  of  Ehrlich.  Both  of  these  are  extremely  ingeni- 
ous and  explain  satisfactorily  why  certain  bacteria  are  unable  to 
infect  the  body,  and  why,  the  body  once  infected,  cannot,  in  many 
diseases,  be  again  infected.  Furthermore  these  theories  make  it 
clear  to  us  why  the  body  tissues  during  life  do  not  fall  an  easy 
prey  to  many  putrefactive  bacteria,  as  after  death. 

Phagocytosis  is  essentially  a  theory  of  cell  devouring.  Leuco- 
ytes  which  are  white  mobile  cells  of  the  blood,  and  other  fixed  cells, 
defend  the  body  against  infection  by  devouring  the  invading  agents 
of  disease.     (Fig.  i6.) 


i 


^^f::i.J^rsr-r^^^-f^-'^ 


PHAGOCYTOSIS  43 

Metchnikoff  considers  the  subject  of  phagocytosis  under  three 
aspects:  i.  Nutritional.  2.  Resorptive.  3.  Protective. 
7^  Nutritional. — Amceba  and  certain  other  unicellular  vegetable 
organisms  belonging  to  the  myxomycetes  possessing  amoeba  proper- 
ties and  having  the  faculty  of  throwing  out  pseudopodia  or  proto- 
plasmic arms,  acquire  their  food  by  enveloping  smaller  organisms, 


Fig.    16. — ^Phagocytosis.     Gonococci  in   leucocytes  in   pus   from   gonorrhoea. 
(KoUe  and  Wassermann.) 

and  other  nutritious  matter  which  they  absorb.  Certain  intracellu- 
lar ferments,  which  they  possess,  digest  fibrin  and  gelatine,  and  con- 
vert starch  into  sugar.  These  cells  protect  themselves  against  inim- 
ical micro-organisms  by  enveloping  and  digesting  them.  \' 
.  They  are  attracted  by  food  and  moisture  (called  positive  chemo- 
taxis)  and  repelled  by  strong  solution  of  salt,  poisons,  etc.  {negative 
chemotaxis).  i 

Higher  in  the  animal  scale  among  the  multicellular  organisms,  the 
cells  of  the  intestines  have  the  property  of  absorbing  and  digesting 
food.  These  fixed  cells  are  called  sessile  phagocytes.  Still  higher 
in  the  scale  (man)  certain  digesting  cells  are  present  in  the  digestive 
tract,  which  are  incapable  of  absorbing  food.  They,  however, 
secrete  ferments  which  digest  gelatine  and  fibrin,  and  convert  starch 


44  IMMUNITY 

into  sugar.  They  are  not  directly  concerned  in  the  nutrition  of  the 
organism.  Cells  of  a  protecting  character  in  man  are  either  micro- 
phages,  or  macrophages.  The  microphages  are  the  polynuclear 
leucocytes,  which  are  concerned  in  the  protection  of  the  organism 
against  acute  infections,  the  bacteria  of  which  they  take  up  and 
devour.  The  macrophages  consist  of  the  large  lymphocytes,  the 
endothelial  cells,  and  some  connective  tissue  cells,  which  take  up 
foreign  bodies.  Both  of  these  classes  contain  ferments;  micro- 
cytase  being  found  in  the  microphages;  and  macrocytase  in  the 
macrophages.  The  latter  absorb  connective  tissue  cells  through 
their  particular  ferments,  and  are  active  in  immunizing  against 
tuberculosis.  These  cells  perform  various  functions  in  the  body. 
When  the  tissues  are  invaded  with  bacteria,  the  blood  shows  an 
increase  in  the  number  of  these  microphages,  which  have  been 
called  the  "hygienic  police.'*  Summoned  to  repel  invasion,  they 
leave  the  lymph  stream  for  that  of  the  blood.  All  the  phenomena  of 
leucocytic  emigration  in  inflammation  is  a  manifestation  of  positive 
chemotaxis.  During  practically  all  the  infections,  the  peripheral 
blood  contains  an  excess  of  leucocytes  over  the  normal  amount  per 
cubic  millimeter  (7,600).  In  exceptional  infections,  typhoid  fever, 
influenza,  measles,  and  tuberculosis,  there  is  no  such  increase,  or 
leucocytosis.  In  malaria  (not  a  bacterial  infection)  there  is  also 
no  leucocytosis. 

Metchnikoff  has  described  a  process  in  which  the  phagocytes 
undergo  what  he  calls  phagolysis.  The  ferment,  cytase,  is  dis- 
charged and  acts  extracellularly,  as  in  the  haemolysis  of  foreign  red 
blood  cells  in  the  peritoneum  of  a  guinea  pig.  This  phagolysis  in 
dissolving  of  the  leucocyte  is  the  cause  of  the  chemical  phenomena 
(so  he  avers)  which  cannot  be  ascribed  merely  to  phagocytosis. 
Metchnikoff  further  claims  that  both  phagocytosis  and  phagolysis, 
either  severally,  or  in  combination,  are  responsible  for  natural  or 
acquired  immunity. 

In  the  case  of  acquired  immunity,  it  is  supposed  that  the  leuco- 
cytes  become    educated.     Regarding    the    toxins    against    which 


PHAGOCYTOSIS  45 

animals  can  be  immunized  by  gradually  increased  doses,  it  is  held 
by  him  that  the  educated  leucocytes  neutralize  the  poison  by  their 
secretions. 

In  the  case  of  anthrax  infection,  animals  infected  with  virulent 
cultures  of  this  organism  quickly  succumb,  without  exhibiting  any 
leucocytosis  (negative  chemo taxis). 

If  the  animal  has  been  previously  immunized  with  attenuated 
culture  the  injection  of  a  virulent  culture  is  followed  by  an  enormous 
outpouring  of  leucocytes  at  the  site  (positive  chemotaxis),  while  if 
the  site  of  the  inoculation  in  the  non-immune  animal  is  examined, 
only  a  few  leucocytes,  and  some  clear  serum  will  be  found. 

Toxins,  if  injected,  cause  a  negative  chemotaxis.  If  tetanus 
spores  are  injected  into  an  animal,  together  with  some  toxin,  the 
animal  rapidly  succumbs  to  tetanus,  without  evincing  any  leuco- 
cytosis. If  the  spores  are  washed  free  from  toxin,  and  injected, 
active  leucocytosis  occurs  and  the  animal  survives. 

A  mixed  infection  of  a  highly  virulent  culture,  and  a  non-virulent 
one,  often  hastens  the  action  of  the  virulent  one.  It  is  supposed 
that  the  non- virulent  bacteria  engage  the  leucocytes,  so  that  these 
cells  cannot  cope  with  the  virulent  ones. 

Phagocytosis  thus  plays  an  important  part  in  the  protection  r6le 
in  natural  immunity,  but  no  satisfactory  theory  has  yet  been  offered 
in  explanation  of  the  protective  process  in  acquired  immunity,  at 
least  against  toxins  and  other  soulble  and  unorganized  poisons. 

In  order  to  meet  the  criticisms  arising  after  Ehrlich's  theories, 
Metchnikoff  added  to  his  theory  by  stating  that  complement  and 
anti-body  are  enzymic  bodies  derived  from  phagocytes. 

The  cellulo-humeral  theory  claims  the  attention  of  most  bacteri- 
ologists, as  the  probable  explanation  of  the  phenomena  of 
immunity. 

It  is  certain  that  cells,  either  sessile  or  mobile,  and  fluids,  are  im- 
portant means  of  internal  defense.  In  order  that  this  theory  may 
be  comprehended,  certain  well  known  properties  of  normal  'and 
artificially  immunized  serum  must  be  understood. 


46  IMMUNITY 

y^  Alexins. — It  has  been  found  by  numerous  observers,  that  normal 
blood  serum  is  germicidal  for  many  bacteria,  and  the  peculiarly 
active  substance  that  is  contained  in  the  serum,  was  called  by  Buch- 
ner  Alexin.     This  dissolves  bacteria  and  destroys  them.     It  also 

.  destroys  the  red  blood  cells  of  other  animals.  The  alexin  of  a  dog's 
serum  dissolves  the  red  cells  of  a  rabbit;  it  is  therefore  hcemolytic. 
It  also  is  thermolabile,  that  is,  its  properties  are  destroyed  by  heat 
(55°  C).  It  is  identical  with  the  complement  of  Ehrlich,  and  the 
cytase  of  Metchnikoff .  K, 

><^^The  complement,  as  it  will  hereafter  be  called,  takes,  as  already 
stated,  an  active  part  in  bacteriolysis,  or  bacteria-dissolving,  and 
in  haemolysis,  or  blood -dissolving,  it  is  present  in  normal  non-im- 
mune sera.  R.  Pfeiffer  found  that  if  some  serum  from  a  guinea  pig 
immunized  against  cholera  spirilla  is  injected  into  the  peritoneal 
cavity  of  a  healthy  non-immune  guinea  pig,  with  some  cholera 
spirilla,  that  the  latter  are  agglutinated,  and  ultimatel;^  dissolved, 
having  undergone  bacteriolysis  (Pfeiffer' s  reaction) ,/^The  immune 
serum  alone  in  a  test-tube,  with  the  cholera  spirilla  does  not  have 
this  action,  but  if  some  normal  guinea  pig  serum  is  added  to  the 
mixture,  an  immediate  solution  takes  place,  showing  that  the 
presence  of  both  the  normal  serum  containing  the  complement, 
and  the  immune  serum,  containing  the  immune  body,  or  am- 
boceptor, are  necessary  to  complete  the  solution  of  the  bacteria. 
If  the  complement  is  heated  above  55°  C.  for  an  hour,  solution 
does  not  take  place,  even  if  the  immune  serum  is  present,  but, 
after  heating  the  mixture,  it  may  be  reactivated  by  adding  some 
fresh  unheated  complement.  The  complement  is  thermolabile  i.e., 
destroyed  by  heatT) 

VJThe  immune  serum  is  not  affected  by  heat,  and  is  theretore  called 
thprmostabilej 
Ijrhese  various  reactions  may  be  expressed  concretely  thus: 

Bacteria -h  immune  body  =  no  solution. 
B  acteria + complement = no  solution. 


SERUM  CHEMISTRY  47 

Bacteria  +  immune  body  +  complement  =  solution  (Pfeiffer's  re- 
action). 

Bacteria + immune  body  +  complement  (heated)  =  no  solution. 
Bacteria + immune  body  (heated)  +  complement  =  solution. 

I  The  same  phenomena  in  the  blood  of  animals  immunized  against 
the  red  blood  corpuscles  of  another  animal  of  foreign  species  have 
been  observedTj 

Llfa  rabbit  is  immunized  with  the  blood  of  a  dog  by  repeated  and 
increasing  doses,  the  serum  of  that  rabbit  will  become  hcdmolytic  to 
the  corpuscles  of  the  dog's  blood  if  they  are  mixed,  provided  some 
normal  rabbit's  blood  complement  is  added  to  the  mixture? 

Dog's  erythrocytes  +  immune  rabbit  serum = no  solution. 

Dog's  erythrocytes  +  immune  rabbit  serum  +  complement = solu- 
tion. 

Dog's  erythrocytes  +  immune  rabbit  serum -f  complement,  heated 
=  no  solution. 

^he  immune  body  acts  as  a  preparer  of  the  corpuscles,  or  bac- 
teria, so  that  the  complement  can  act  upon  the  cells.  The  reaction 
is  very  like  the  action  of  pepsin  on  fibrin.  Hydrochloric  acid  must 
be  present^ 

(i)  Pepsin  +  fibrin = no  solution  or  lysis. 

(2)  HCH- fibrin = no  solution  or  lysis. 

(3)  Pepsin +  HCl-f  fibrin  =  solution  or  lysis. 
The  HCl  corresponds  to  the  immune  body. 

In  the  case  of  haemolysis,  or  bacteriolysis  the  action  of  the  immune 
body  is  specific.  (The  immune  body  of  cholera  spirilla  will  not 
prepare,  or  fix  typhoid  bacilli,  so  that  they  can  be  acted  upon 
by  the  complement.  Nor  will  the  immune  body  of  dog's  ery- 
throcytes prepare  these  of  a  pigj  so  that  the  complement  may 
act  on  it. 


48  IMMUNITY 

A  loose  chemical  union  takes  place  between  the  bacteria  and  the 
immune  body,  but  no  such  union  occurs  between  the  complement 
and  the  bacteria.  The  same  chemical  union  occurs  between  the 
red  cells  and  the  immune  body  in  haemolysis,  but  not  between  the 
cells  and  the  complement. 

Ehrlich  holds  that  there  are  many  complements,  each  one  different 
from  the  other,  and  that  their  action  is  specific  for  the  different  kinds 
of  bacteria  or  cells  with  which  an  animal  may  be  immunized.  Bor- 
det  and  Buchner,  on  the  other  hand,  maintain  that  there  is  but  one 
complement. 

The  solution  of  any  cells  by  immune  bodies,  or  anti-bodies,  as 
they  have  been  called,  is  known  as  cytolysis.  And  cytolysins  may 
be  produced  by  making  anti-bodies  of  nerve  cells,  leucocytes,  epithe- 
lial cells,  liver  cells,  as  well  as  blood  cells,  by  immunizing  an  animal 
against  these  different  cells  with  repeated  injections  of  the  cells  or 
emulsions  of  them. 

^Agglutinins  are  peculiar  bodies  which  have  the  property  of  caus- 
ing certain  cells  to  agglutinate.  One  of  the  earliest  manifestations 
of  immunity  of  a  certain  serum  to  bacteria,  or  to  blood  cells,  is  this 
peculiar  action  of  the  serum  causing  either  the  bacteria  or  blood 
cells  to  clump  together  in  masses.  Part  of  Pfeiffer's  reaction  is  the 
agglutination  of  the  cholera  spirilla  in  clumps  before  they  are  dis- 
solved by  the  complement  and  immune  body. 

If  the  serum  of  a  typhoid  fever  patient  is  mixed,  even  in  high  dilu- 
tions with  some  typhoid  bacilli,  the  latter  are  clumped  in  isolated 
groups.  Clinically  this  is  known  as  the  Widal  reaction,  and  is  the 
most  reliable  single  sign  of  typhoid  fever. 

These  agglutinins  may  be  produced  artificially  by  injecting  large 
and  increasing  doses  of  bacteria  into  animals.  After  a  time,  in 
the  serum  of  the  rabbit,  there  develops  a  peculiar  body  which 
agglutinates  typhoid  bacilli,  if  they  are  brought  in  contact  with  it. 
Sera  can  be  rendered  so  highly  agglutinative  as  to  produce  this 
reaction  even  if  diluted  100,000  times  or  more. 

If  an  animal  is  immunized  against  spermatozoa,  or  the  red  blood 


PRECIPITINS  49 

cells  of  a  foreign  species,  its  serum  becomes  agglutinative  to  these 
cellsT^ 

[[Precipitins. — If  a  rabbit,  or  any  other  animal  in  fact,  is  immu- 
nized by  repeated  injections  of  blood  foreign  to  it,  peculiar  bodies 
develop  in  its  blood  serum  called  precipitins,  and  these  can  be 
demonstrated  by  adding  to  the  serum  of  the  immunized  animal 
in  a  test-tube  a  minute  portion  of  the  blood  against  which  the 
animal  was  immunized.  As  soon  as  the  immunized  serum  and  the 
specific  blood  are  mixed,  a  precipitate  forms.  This  is  another 
phenomenon  of  immunity,  and  is  of  more  than  theoretical  import- 
ance in  medicine.  The  reaction  is  strictly  specific ;  thus,  if  the  serum 
of  a  goat  is  injected  into  a  rabbit  repeatedly  the  rabbit's  blood  will 
form  a  precipitate  with  normal  goat's  serum  if  the  two  are  mixed 
in  a  test-tube.  Old  dried  blood,  semi-putrid  blood,  blood  on  white- 
wash, or  rusty  steel,  even  in  minute  quantities,  if  dissolved  in  salt 
solution,  may  be  used  to  produce  this  reaction.  In  medico-legal 
matters,  this  test  is  of  use  for  the  identification  of  human  blood. 
Naturalists  also  use  this  method  for  the  differentiation  of  species. 
By  many,  the  phenomenon  of  agglutination  is  supposed  to  be  due 
to  the  formation  of  a  precipitin,  in  the  meshes  of  which  bacteria  or 
blood  cells  are  caught  and  agglutinated,  and  that  agglutination  is 
but  a  modification  of  the  formation  of  precipitins. 

Anti-toxin  formation  is  also  another  phenomenon  of  immunity. 
If  an  animal,  such  as  a  horse,  receives  numerous  increasing  doses 
of  a  given  toxin,  say  that  of  tetanus,  it,  in  a  short  time,  becomes  so 
accustomed  to  the  poison,  that  it  can  withstand  the  administration 
of  immense  doses.  (If  these  large  doses  had  been  given  at  first,  they 
would  have  proved  fatal.)  If  the  horse  is  then  bled,  and  its  serum 
injected  into  rabbits  or  guinea  pigs,  they  may  receive  shortly  after, 
at  one  dose,  enough  toxin  to  kill  ten  such  animals.  The  horse 
serum  thus  protected  these  animals  against  the  toxin,  as  it  was  anti- 
dotal, or  in  other  words  anti-toxic.  A  chemical  union  occurs 
between  the  toxin  and  the  anti-toxin,  since,  according  to  the  law  of 
multiples,  a  definite  amount  of  anti-toxin  unites  with  a  definite 
4 


50  IMMU.NITY 

amount  of  toxin.  If  ten  times  the  amount  of  anti-toxin  is  used  it 
will  exactly  neutralize  ten  times  the  amount  of  toxin,  and  the  mix- 
ture becomes  inert.  Again,  the  union  of  the  two  substances  follows 
well  known  chemical  laws,  whereby  chemical  union  takes  place 
more  rapidly  in  concentrated  than  in  dilute  solutions,  and  when  the 
solutions  are  warm.  If  the  mixture  of  toxin  and  anti-toxin  is  heated, 
it,  instead  of  being  neutral,  becomes  toxic  again.  This  toxicity  can 
be  neutralized  again  by  the  addition  of  fresh  unheated  anti-toxic 
serum  {reactivation) . 

The  production  of  bacteriolysins,  cytolysins,  agglutinins,  precipi- 
tins, and  anti-toxins  are  manifestations  of  the  activity  of  the  immun- 
ized organisms.  To  further  understand  this  activity,  Ehrlich's 
side-chain  theory  of  immunity  must  be  comprehended.  This  is 
known  as  the  chemical  theory.  To  understand  it  fully  some  con- 
sideration must  be  given  to  the  study  of  the  toxin  molecule.  Ehr- 
lich  believes  that  each  molecule  of  toxin  is  made  up  of  two  groups 
of  atoms,  constituting  what  is  known  in  chemical  nomenclature  as 
lateral  chains. 

Many  molecules  are  made  up  of  a  central  body  and  lateral  chain 
of  atoms  which  are  free  to  combine  with  other  groups  of  atoms  with- 
out disturbing  the  central  body. 

The  benzol  ring  is  very  suitable  for  the  demonstration  of  the 
relationship  of  the  side-chain  to  the  central  body. 

H 

I 

H— C-^   "^C— H 

II  I 

H— a       ^C— H 

I 

H 

BENZOL. 

The  benzol  molecule  C^Hg  is  here  represented  graphically  as  a 


LATERAL  CHAIN  THEORY  5 1 

ring  with  a  central  nucleus  of  Cg  with  lateral  chains  of  H.  con- 
necting each  atom  of  C. 

If  one  of  these  lateral  chains  H.  is  supplanted  by  the  acid  radical 
CO  OH.  the  benzol  is  converted  into  benzoic  acid  and  its  formula  is 
represented  thus: 

O  - 

// 
C— OH 

! 

H— C^    ^C— H 

II  I 

H — Cv       ^C — H 

I 
H 

BENZOIC  ACID. 

If  to  this  acid  radical  of  the  benzoic  ring,  sodium  hydroxid  unites, 
supplanting  an  H  in  the  OH  of  this  radical,  we  have,  instead  of 
benzoic  acid,  benzoate  of  soda. 

O 

// 
C— O— Na 

/\ 
H— C       C— H 

II         I 
H— C       C— H 

\^ 

c  : 

I 

H 

BENZOATE  OF  SODA. 

It  is  thought  that  as  the  soda  is  brought  in  contact  with  the  central 
nucleus  of  the  benzol  ring,  so  food  stufiFs  unite  with  the  central  body 
of  the  cell  molecule  in  the  organism  and  nourish  it. 


52  IMMUNITY 

In  the  case  of  toxin,  the  two  lateral  chains  of  its  molecule  are 
called  haptophores  and  toxophores.  The  haptophores  seize  the 
lateral  chains  of  the  cell  and  the  toxophores  poison  it. 
(  Ehrlich  conceived  that  cells  were  nourished  by  their  lateral  chains, 
each  having  a  central  nucleus  with  many  lateral  chains  called  recep- 
tors bristling  all  over  it.  Complex  albumins,  food  stuffs  or  poisons 
(as  the  case  may  be)  unite  with  it.  This  means  a  chemical  union  of 
a  part  of  a  cell  with  all  or  part  of  a  group  of  atoms.  But  certain 
body  cells  are  only  capable  of  uniting  with  certain  toxins.  It  is 
known  that  the  toxin  of  tetanus  has  a  chemical  affinity  for  the 
nervous  system  and  for  its  neural  elements  and  not  for  liver  or 
spleen  cellsj 

Q]he  poisons  of  snake  venom  seem  incapable  of  uniting  with 
any  cells  of  the  pig;  this  animal  is,  therefore,  immune  to  snake 

venom.3 

^Urow,  as  these  toxins  unite  with  the  cells  by  means  of  the  receptors, 
the  cell  is  stimulated  to  produce  an  excessive  number  of  these  recep- 
tors, which  are  cast  off  and  become  free.  Nature  is  very  prodigal 
and  whenever  any  of  the  tissues  of  the  body  have  been  injured,  or 
there  is  a  deficiency,  an  enormous  excess  of  reparative  cells  is  pro- 
duced^  Weigert  first  called  attention  to  this  phenomenon,  which 
has  been  called  Weigert' s  over-production  theory,  l^o  when  the 
haptophores  of  the  toxin  molecule  combine  with  the  receptors  of  the 
cell,  the  latter  are  incapable  of  any  further  union  and  are  useless  to 
the  ceff\  Accordingly  a  great  number  of  free  receptors  are  gener- 
ated, and  floating  in  the  blood,  engage  the  haptophorous  portion  of 
the  toxin.  Thus  the  toxophore  is  neutralized  and  rendered  innocu- 
ous before  it  can  reach  the  cell.  These  free  over-produced  recep- 
tors constitute  the  anti-toxin.  This  is  the  essence  of  Ehrlich's 
theory.     (Fig.  17.) 

Through  the  process  of  time  and  oxygenation,  the  toxophorous 
group  in  the  toxin  becomes  innocuous,  and  only  the  haptophorous 
group  remains  active;  nevertheless  the  haptophorous  group  is  able 
to  combine  with  the  receptors  and  to  stimulate  the  cell  into  generat- 


RECEPTORS 


53 


ing  free  receptors.  This  attenuated  toxin  is  called  by  Ehrlich 
toxoid.  The  receptors  have  been  compared  to  a  lightning  rod, 
which  if  placed  within  a  building  would,  if  struck,  cause  disaster, 
while  the  same  rod  placed  outside  of  the  building,  is  a  means  of  pro- 
tection to  the  structure  against  lightning.  This  theory  can  be 
applied  to  the  production  of  other  anti-bodies.  If  blood  cell, 
bacterial  cell,  or  any  animal  fluid  possessing  a  haptophore  is  capable 
of  combining  with  side-chains  (receptors)  of  the  cells  of  the  immu- 


FiG.  17. — a,  receptor  on  cell;  b,  toxin  molecule;  c,  haptophorous  portion  of 
the  molecule;  J,  toxophorous  portion;  e,  receptor.     (Williams.) 


nized,  just  as  a  key  fits  a  lock,  then  the  cells  are  stimulated  to  pro- 
duce excessive  numbers  of  receptors,  and  these  constitute  the  anti, 
or  immune  body.  It  is  possible  to  produce  from  rennet,  egg-albu- 
min, cow's  milk,  and  from  many  other  albuminous  substances, 
immune  bodies  by  injecting  these  substances  into  animals.  (Figs. 
18,  19.) 


54 


IMMUNITY 


Fig.  i8.— EHRLICH'S  LATERAL  CHAIN  THEORY.  Cell  with  numer- 
ous receptors  of  various  kinds  and  shapes  to  which  are  united  the  toxin  mole- 
cule.    Note  the  free  receptors. 


v!'  rMi 


; 


Fig.  19.— EHRLICH'S  LATERAL  CHAIN  THEORY.  In  one  figure  the 
free  receptors  (anti-bodies)  are  united  with  the  toxin  molecule,  the  attached  re- 
ceptors have  no  haptophores  united  to  cell. 


IMMUNE  BODIES 


55 


List  of  immune  bodies  and  their  anti-bodies  (Ricketts) . 
Antigens  or  Products  of 

Immunizing      Immunization 
Substances 


Toxins 

Anti-toxins 

Complements 

Anti-comple- 
ments 

Ferments 

Anti-ferments 

Precipitogenous 

Precipitins 

Substances 

Agglutinogenous 

Agglutinins 

Substances 

(  Hemolysins 

Opsinogenous 

Opsinins 

Bacteriolysins 

Substances 

Special  cytotoxins 

Consisting    oj 

Cytotoxin  Produc- 

Cytotoxins  .  . 

Such  as 

two  bodies 

.  ing  Substances 

Spermotoxin 

Complement 

Nephrotoxin 

Ambocepter 

Hepatotoxin,  etc. 

Syno 

nyms. 

Complement 

Ambocepter. 

Alexin. 

Immunkorper. 

Cytase. 

Zwischenkorper. 
Intermediary  body. 
Fixaleur. 
Preparateur. 
Copula. 

Desmon. 
Substance  sensibilis 

atrice. 

It  is  well  known  that  rennet  coagulates  milk,  but  if  some  of  the 
serum  of  an  animal  immunized  against  rennet  is  added  to  the  milk, 
the  latter  cannot  be  coagulated  because  the  anti-rennin  combines 
with  the  rennet  and  renders  it  inert. 


56  IMMUNITY 

The  production  of  bacteriolysins  is  explained  by  Ehrlich's  lateral- 
chain  hypothesis.  Immunization  against  bacteria  which  do  not 
produce  soluble  toxins  is  easily  secured  by  repeated  injection  of 
either  dead  or  living  bacteria  into  the  organism.  It  is  not  easy,  how- 
ever, to  confer  passive  immunity,  as  in  the  case  of  diphtheria,  by 
the  injection  of  the  serum  of  the  immunized  animal.  The  im- 
mune body  is  alone  present  in  the  serum  generally  and  some  com- 
plement must  be  added  to  effect  bacteriolysis.  The  serums  which 
aid  in  the  solution  of  bacteria  are  known  as  anti-bacterial  ser- 
ums, which,  though  not  anti-toxic,  may  check  invasions  and  aid  in 
recovery  by  destroying  bacteria.  It  is  possible  to  effect  an  in  cor- 
pore  bacteriolysis  in  the  case  of  typhoid  fever  if  the  immune  body 
and  complement  are  injected  in  sufficient  amounts  and  proportions. 
As  yet  the  results  are  not  satisfactory  from  a  clinical  standpoint. 

A  study  of  figure  20  will  show  clearly  the  exact  combinations  of 
various  substances  engaged  in  the  immunity  process.  Some  of  the 
terms  must  be  defined. 

Antigen,  the  body  bacterium;  red  blood  cell,  etc.,  used  for  stimu- 
lating the  production  of  thermostabile  anti-bodies,  which  latter  are 
then  the  substances  formed  against  antigens;  inciting  substance- 
antigen. 

Toxins,  ferments,  see  above. 

Toxophore,  the  poison-carrying  fraction  of  the  antigen. 

Haptophore,  the  binding  fraction  of  antigen  or  anti-body. 

Complement,  the  normal  thermolabile  anti-body  substance  in 
serum. 

Zymophore,  toxophore  for  agglutinins  and  precipitins. 

Cytophile  fraction  is  that  part  of  anti-body  which  combines  with 
cell,  while  complementophile  fraction  joins  with  complement. 

Immune  body,  the  thermostabile  anti-body  against  bacterial  or 
other  cells. 

By  immunizing  with  complement  or  antibody  we  obtain  respect- 
ively anti-complement  and  anti-immune  body  which  experimentally 
will  neutralize  the  action  of  these  two  substances.     The  comple- 


58  IMMUNITY 

ment  being  the  really  responsible  potent  factor  in  all  these  reactions 
it  may  be  assumed  to  have  two  binding  affinities,  one  to  the  cells 
which  it  designs  to  help  and  another  effect  upon  antigen.  If  the 
former  be  absorbed  in  any  abnormal  manner  the  latter  is  valueless. 

Cell  Receptor  and  Immune  bodies  (follow  figure  20).  First  Order: 
Simple  union  of  toxins  (soluble)  and  fixed  or  free  receptors  or  anti- 
toxins; no  complement  needed. 

Second  Order:  Concerns  agglutination  and  precipitation.  Anti- 
gen has  two  affinities,  one  for  the  haptophore  of  anti-body,  another 
for  the  agglutinin  of  the  anti-body.  The  anti-body  must  therefore 
have  reversed  corresponding  fractions.  The  zymophore  of  anti- 
body acts  when  the  two  haptophores  have  united  and  produced  the 
agglutination  or  precipitation.     No  complement  is  needed. 

Third  Order:  Concerns  bacteriolysins,  hemolysins  or  bacteroly- 
sins,  etc.;  have  haptophore  for  anti-body,  and  a  toxophore.  Anti- 
body has  haptophore  for  antigen  and  for  the  haptophore  of  the 
complement.  The  union  of  the  three  must  occur.  Complement  is 
necessary  for  the  destruction  of  the  bacteria  which  it  accomplishes 
through  its  zymophore. 
-y^  Anaphylaxis. — Against  protection,  the  opposite  of  prophylaxis; 
also  called  Hypersusceptibility.  This  phenomenon,  first  described 
by  Theobald  Smith,  Portier  and  Richet,  consists  in  a  condition  of 
extreme  sensitiveness  of  animals  against  foreign  proteins.  If  a 
guinea  pig  be  injected  into  the  peritoneum  with  a  minute  quantity, 
say  j^nnr  ^^  ^  gi'^-ni,  of  horses'  serum  and  eight  to  ten  days  later 
receive  a  quantity  of  ^^  of  a  gram,  the  animal  will  become  uneasy, 
then  depressed,  have  dyspnea,  scratch  itself  violently  about  the 
face  and  finally  die  after  an  intensification  of  these  symptoms. 
Similar  symptoms  have  been  observed  in  persons  receiving  diph- 
theria anti-toxin  therapeutically.  The  condition  of  high  sensitivity 
to  this  anti-toxin  is  called  allergie  and  upon  its  degree  depends  the 
reaction  following  anti-toxin  administration.  The  skin  eruptions, 
joint  pains  and  edema  of  serum  sickness  are  also  evidences  of  this 
condition.     It  is  said  that  those  persons  who  suffer  after  anti-toxin 


ANAPHYLAXIS  59 

are  susceptible  to  the  emanations  from  horses.  This  wilJ  not 
explain  all  cases  however.  The  scientific  world  is  beginning  to 
consider  the  contraction  of  any  infectious  disease  as  an  evidence  of 
anaphylaxis  on  the  patient's  part  to  the  causative  agent. 

In  experimentally  induced  hypersusceptibility  the  reaction  is 
specific.  The  condition  is  transmissible  from  mother  to  fetus 
and  it  can  be  transferred  from  adult  to  adult  passively  by  injecting 
the  blood  of  a  sensitive  animal  into  a  normal  one.  The  first  dose 
is  called  the  sensitizing  one,  the  second  the  intoxicating.  The 
incubation  period  of  the  sensitization  varies  with  the  nature  of  the 
protein;  for  horse  serum  it  is  from  eight  to  twelve  days,  for  bacterial 
proteins  from  five  to  eight  days.  The  sensitive  period  may  last 
for  several  years.  In  searching  for  the  cause  of  this  reaction  it  was 
found  that  there  are  (i)  a  spastic  distention  of  the  pulmonary  alveoli 
probably  both  of  central  and  local  nature,  (2)  scattered  hemorrhages 
in  the  organs  and  (3)  hemorrhages  with  ulcerations  in  the  gastric 
mucosa.  There  have  been  many  theories  for  this  phenomenon, 
but  those  of  Vaughan,  Friedberger  and  Wolff  Eisner  may  be  con- 
densed and  compounded  about  as  follows.  The  body  is  unprepared 
to  care  for  parenterally  (otherwise  than  gastrointestinal  tract) 
introduced  protein  and  must  develop  an  anti-body  or  enzyme  to 
care  for  it.  This  enzyme  or  anti-body  works  slowly  and  carefully 
disposes  of  the  foreign  protein,  the  products  of  which  are  slowly 
absorbed  and  removed.  In  accord  with  the  overproduction  theory 
this  anti-substance  is  in  large  quantity  when  another  introduction  of 
protein  occurs,  and  goes  to  its  work  with  avidity  so  that  it  rapidly 
breaks  the  protein  up  into  toxic  elements  which  cannot  suddenly 
be  cared  for  by  the  body.  These  protein  toxins  attack  nervous  and 
parenchymatous  tissues. 

It  has  been  shown  that  an  anti-anaphylactic  state  can  be  pro- 
duced by  repeated  small  injections  of  protein  at  intervals  too  short 
to  allow  incubation  of  an  intoxicating  dose. 

Friedberger  has  used  these  facts  to  elaborate  a  theory  of  infection. 
He   believes  that  bacteria  circulating  in  the  body  stimulate  anti- 


6o  IMMUNITY 

bodies,  combine  with  them  and  that  when  complement  acts  upon 
this  union  toxic  substances  are  set  free. 

In  explaining  all  infectious  diseases  on  this  basis  one  assumes 
that  sometime  in  life  a  person  has  been  sensitized  by  bacteria  or 
their  proteins  so  that  he  is  receptive  for  a  virulent  germ  when 
this  has  overcome  the  primary  external  bodily  defenses.  It  is  also 
to  be  considered  the  modern  explanation  of  diatheses. 

McKail  divides  anaphylaxis  as  follows: 

Natural  Anaphylaxis,  depending  upon 

a.  Species  of  animal,  for  example  cholera  in  man,  anthrax 
in  cattle,  glanders  in  horses. 

b.  Age — diphtheria  in  children,  erysipelas  in  the  elderly. 

c.  Individual — to  white  of  egg,  or  blood  serum,  even  by  inges- 
tion ("one  man's  meat  is  another  man's  poison"). 

Acquired  Anaphylaxis,  depending  upon 

a.  An  attack  of  disease,  erysipelas,  diphtheria. 

b.  The  injection  of  dead  cells,  tuberculin. 

c.  Injection  of  nitrogenous  matter,  blood  serum  and  egg-white. 

/  Complement  Fixation. — Hemolysis  occurs  when  the  serum  of 
a  rabbit  immunized  against  washed  sheep's  red  blood  cells  is  mixed 
with  fresh  washed  sheep's  corpuscles  in  the  presence  of  complement. 
If,  however,  complement  be  absorbed  in  any  way  a  solution  of  the 
coloring  matter  of  the  red  cells  will  not  occur  in  this  mixture. 
Complement  will  combine  with  anti-body  in  the  presence  of  antigen. 
This  fact  has  been  taken  advantage  of  in  determining  both  the 
nature  of  antigen  and  the  presence  of  anti-body.  Its  most  important 
practical  use  is  in  syphilis,  to  the  diagnosis  of  which  Wassermann 
applied  it,  and  the  Wassermann  test  is  for  the  presence  of  syphilitic 
anti-body  in  the  blood  serum  of  syphilitics.  This  test  is  positive 
from  the  initial  lesions  all  during  life  unless  the  patient  has  been 
successfully  treated.  Indeed  the  parasyphilitic  states  also  give  it. 
The  principles  of  the  test  are  also  used  for  determining  the  pres- 
ence of  tuberculous,  leprous,  typhoid  and  other  anti-bodies. 


WASSERMAN  REACTION 


6l 


The  materials  necessary  in  the  Wassermann  test  are  as  follows: 

I.  Syphilitic  antigen,  extract  from  the  syphilitic  liver  of  a  fetus, 
in  alcohol,  ether  or  water;  lipoids  like  lecithin  or  extracts  from 
guinea  pig's  heart  are  said  to  act  as  antigen. 

2a.  Serum  from  a  known  case  of  syphilis  and  containing  therefore 
syphilitic  antibody. 

2b.  Known  non-syphilitic  serum  without  anti-body. 

3.  The  suspected  serum. 

4.  Fresh  serum  from  a  guinea  pig,  rich  in  complement. 

5.  Serum  from  a  rabbit  that  has  been  immunized  against  washed 
red  cells  from  a  sheep;  called  amboceptor. 

6.  Fresh  washed  sheeps'  red  blood  cells. 

The  solutions  are  all  standardized  so  that  only  sufficient  of  each 
is  added  to  complete  the  absorption  or  produce  the  hemolysis.  The 
serum  known  to  be  syphilitic  and  the  suspected  serum  are  heated 
to  56°  C.  for  30  minutes  to  destroy  the  native  and  inherent  comple- 
ment.   The  rabbit  anti-sheep  cell  serum  is  also  heated  to  this  degree. 

The  hemolytic  series,  i.e.,  sheep's  cells,  rabbit's  anti-sheep's  cells, 
serum  and  complement  are  standardized  to  find  out  what  quantities 
will  exactly  complete  hemolysis.  These  quantities  are  the  units. 
It  is  necessary  in  control  tests  to  find  out  what  quantity  of  the  antigen 
and  known  syphilitic  anti-body  will  unite  to  bind  the  determined 
quantity  of  complement.  The  tests  are  performed  in  small  tubes 
so  as  to  have  a  long  column  of  fluid  easier  to  observe.  Tubes  are 
set  as  follows: 


unit  #1 
unit  #] 


+  I  unit  #2a+  I  unit  #4. 
+  I  unit  §3  +  1  unit  §4. 


C.  I  unit  #1  +  1  unit  #2b  + 

D.  I  unit  #2a+  i  unit  §4. 

E.  I  unit  #2b-|- 1  unit  #4. 

F.  I  unit  #3+1  unit  §4. 

G.  I  unit  #4. 
H.  1  unit  #1. 
J.   I  unit  #2a. 
K.  I  unit  #2b. 
L.  •!  unit  #3. 


.  +  I  unit  #5+1  unit  #6  =  No  hemolysis. 
+  I  unit  #5  +  1  unit  #6  = 

if  #3  be  Syphilitic,  no  hemolysis. 

if  #3  be  Non-syphilitic  hemolysis. 
+  I  unit  #5  +  1  unit  #6 »=  Hemolysis. 
-f- 1  unit  #5  +  1  unit  #6  =  Hemolysis. 
+  I  unit  #5  +  1  unit  #6  =  Hemolysis. 
+  1  unit  #5  +  1  unit  #6  =  Hemolysis. 
+  I  unit  #5  +  1  unit  #6  =  Hemolysis. 
+  1  unit  #5+1  unit  #6  =  No  hemolysis. 
+  I  unit  #5+1  unit  #6  =  No  hemolysis. 
+  I  unit  #5+1  unit  #6  =  No  hemolysis. 
+  I  unit  #5+1  unit  #6  =  No  hemolysis 


62  IMMUNITY 

The  tubes  receive  first  the  solutions  on  the  left  and  are  placed 
in  the  37°  C.  incubator  for  2  hours  to  allow  union  of  their  various 
parts,  particularly  the  complement  with  others.  They  then  receive 
the  solutions  on  the  right,  are  placed  in  the  incubator  for  half  an 
hour  and  in  the  ice-box  overnight,  when  they  are  examined  for  a 
.solution  of  the  red  coloring  matter.  If  it  occurs,  the  column  is 
perfectly  clear  red  with  some  residue  of  extracted  cells.  If  no 
hemolysis  has  occurred,  the  red  cells  form  a  layer  at  the  bottom, 
and  the  column  is  clear  and  colorless. 

A  and  B  are  the  tests  of  syphilitic  sera  while  the  remaining  are 
to  find  out  if  the  other  solutions  affect  the  results  of  A  and  B.  Of 
course  tube  G  represents  simply  the  complete  hemolytic  system. 
The  extra  tests  are  to  exclude  the  possibility  of  interference  on  the 
part  of  any  single  member  with  the  complement  No.  4.  The 
character  of  the  test  is  found  in  tube  A  where  syphilitic  antigen  and 
serum  have  bound  or  fixed  the  complement  so  that  it  cannot  unite  with 
the  rabbit  serum  and  sheep'' s  corpuscles  to  hemolyze  the  latter.  This  is 
a  positive  test.  A  negative  test  is  when  hemolysis  occurs^  since  no  anti- 
body is  present  to  unite  with  complement  in  the  presence  of  antigen. 

Complement  Deviation. — This  is  a  condition  arising  when 
there  is  too  much  amboceptor  and  too  little  complement.  The 
free  amboceptors  adsorb  complement  and  there  is  none  left  for  cell 
needs  or  renewed  demands.  It  is  to  be  distinguished  from  com- 
plement fixation.     The  terms  are  not  interchangeable. 

Cholera  and  typhoid  organisms  do  not  produce  soluble  toxins  in 
the  body,  but  when  they  are  disintegrated  therein,  soluble  poisons 
(intracellular)  are  liberated. 

Bacteria  may  become  accustomed  to  the  fluids  of  the  body  by  a 
similar  process  and  may  elaborate  free  receptors  for  their  own  pro- 
tection, i.e.,  anti-bacteriolysins.     (Welch's  theory.) 

In  the  aged,  and  in  chronic  disease  of  the  liver  and  kidneys,  the 
complement  existing  in  the  blood  may  become  reduced  in  quantity, 
and  the  individual  may  succumb  to  an  infection,  which  ordinarily 
would  be  mild. 


MANUFACTURE  OF  ANTI-TOXINS  6^ 

ANTI-TOXINS,  VACCINES,  AND  TOXINS. 

The  following  is  Wassermann's  list  of  anti- toxins: 

Anti-toxins  for  bacterial  toxins  : — 

Diphtheria 

Tetanus 

Botulism 

Pyocyaneus 

Symptomatic  Anthrax 

Anti-leucocidin,  an  anti-toxin  against  the  leucolytic  poison  of 

staphylococcus 
Anti-toxins  for  the  blood  dissolving  toxins  of  certain  bacteria. 

Anti-toxin  for  animal  toxins  : — 

Anti-venene  for  snake  venom 
Anti-toxin  for  spider  poison 
Anti-toxin  for  scorpion  poison 

Anti-toxins  for  certain  poisons  in  fish,  eel,  salamander,  turtle, 
and  wasp  sera. 

Anti-toxins  for  plant  poisons  : — 

.  Anti-ricin  for  castor-oil  poison 
Anti-abrin  for  jequerity  bean  poison 
Anti-robin  for  locust  bean  poison 
Anti-crotin  for  croton-oil  bean  poison 
Anti-pollen  for  pollen  of  plants  that  produce  hay-fever. 

Manufacture  of  Anti-toxins. — If  small  doses  of  a  given  poison, 
such  as  diphtheria  toxin,  be  repeatedly  injected  into  a  susceptible 
animal,  and  if  the  dose  is  gradually  increased,  there  appears,  after 
a  time,  in  the  blood  serum,  an  anti-body,  or  anti-toxin.  This 
substance  in  the  serum  is  secreted  by  the  cells  and  corresponds  to 
the  free  receptors  in  Ehrlich's  lateral-chain  theory.  If  an  animal 
be  injected  with  the  anti-toxin,  and  then  with  a  large  dose  of  toxin — 
say  ten  times  the  amount  necessary  to  kill  it  if  it  had  not  received 
the  anti-toxin — it  will  not  be  harmed.     Here  the  free  receptors  arti- 


64  IMMUNITY 

ficially  supplied  to  the  animal  unite  with  the  haptophorous  chains  in 
the  toxin  molecule,  and  neutralize,  or  bind,  the  toxophorous  or  pois- 
onous chains  in  the  molecule,  and  prevent  toxophore  from  attacking 
important  vital  cells  belonging  to  the  animal.  But  if  the  anti- toxin 
and  toxin,  after  being  mixed  in  a  test-tube,  are  injected  into  a  sus- 
ceptible animal,  no  harm  results,  if  they  are  in  proper  proportions, 
since  the  same  thing  has  happened  in  vitro  that  happened  in  the 
animal,  the  receptors  and  haptophores  have  united;  the  toxophores 
are  bound,  and  the  animal  is  unharmed. 

The  manner  of  making  the  diphtheria  anti-toxin  can  be  taken  as  a 
type. 

Diphtheria  bacilli  are  grown  for  several  days  in  dextrose  bouil- 
lon at  37°  C;  as  the  bacilli  grow  they  elaborate  a  very  powerful 
poison  or  toxin,  which  is  highly  complex  in  compositon.  It  is 
easily  decomposed  by  heat,  light  and  oxygen,  and  should  be  used 
soon  after  it  is  prepared.  After  the  cultures  have  grown  for  several 
days,  the  bouillon  is  filtered  through  a  porcelain  or  Berkefeld 
filter,  and  is  then  stored  in  sterile  bottles  in  an  ice  chest.  Horses  are 
generally  immunized,  since  they  are  susceptible  to  the  action  to  the 
toxin,  and  are  easily  managed.  Before  being  used  they  are  care- 
fully tested  with  tuberculin  for  tuberculosis  and  with  mallein  for 
glanders.  Being  very  susceptible  to  infection  with  tetanus  while 
undergoing  treatment,  a  prophylactic  injection  of  tetanus  anti-toxin  is 
given  each  animal.  McFarland  found  that  the  death  rate  from 
tetanus,  in  a  large  stable,  was  greatly  reduced  after  using  tetanus 
anti-toxin  as  a  prophylactic  measure. 

To  make  anti-toxin,  a  very  virulent  toxin  is  employed.*  A  horse, 
previously  examined  for  health,  is  injected  with  from  .i  to  i.  c.c.  of 
toxin.  This  is  followed  by  a  rise  of  temperature,  local  reaction,  and 
systemic  disturbance.  After  waiting  for  all  reactions  to  disappear 
a  second  injection  is  given,  which  is  followed  by  others  larger  in  size, 
until,  after  a  few  weeks  or  months,  i,ooo  c.c.  of  toxin  are  injected 
at  one  time  (enough  to  have  killed  a  dozen  horses  that  had  not  re- 
ceived the  smaller  doses  previously).     The  injection  of  the  toxin  is 


ANTI-TOXINS  65 

followed  by  an  immediate  fall  in  the  anti-toxic  power  of  the  serum, 
only  to  be  followed  by  a  quick  rise.  The  horse  will  not  produce 
anti-toxin  indefinitely.  After  the  animal  has  been  immunized  suf- 
ficiently, his  blood  is  drawn  from  the  jugular  vein,  and  after  the  clot 
has  formed  the  serum  is  drawn  off  and  stored. 

McFarland  found  that  a  horse  was  capable  of  producing  enough 
anti-toxin  to  protect  806  other  horses  against  doses  of  toxin,  each  one 
of  which  was  equivalent  to  the  total  amount  of  toxin  that  the  immun- 
ized horse  received.  Thus  there  is  evidently  a  tremendous  over- 
production of  anti-toxin  far  above  the  needs  of  the  animal. 

The  various  component  parts  of  the  toxin  stimulate  the  cells  of 
the  horse  to  produce  the  receptors,  or  anti-toxin.  The  toxoids, 
themselves  not  poisonous,  have  the  property  of  stimulating  the  pro- 
duction of  anti-toxin.  We  measure  the  anti-toxic  powers  of  the  anti- 
toxin with  units  arbitrarily  devised.  An  anti-toxic  unit  is  ten  times 
the  least  amount  of  anti-toxic  serum  that  will  protect  a  guinea  pig  weigh- 
ing 300  grams  {standard)  against  ten  times  the  least  certainly  fatal 
dose  of  diphtheria  toxin. 

To  standardize  anti-toxin,  we  must  employ  animals,  into  the 
bodies  of  which  toxins  and  anti-toxins  are  injected.  If  a  certain 
amount  of  anti-toxin  is  necessary  to  protect  a  guinea  pig  against  ten 
times  the  minimum  fatal  dose  of  toxin  per  100  grams  of  guinea  pig 
weight,  then  we  know  that  the  anti-toxin  contains  so  many  units.  A 
unit  dose  of  toxin  is  the  smallest  amount  of  toxin  necessary  to  kill  a 
guinea  pig  weighing  300  grams,  or  the  dose  per  100  grams  of  guinea  pig 
necessary  to  kill. 

Ehrlich's  method  of  standardizing  is  to  obtain  an  an ti- toxin  of 
known  strength  (anti-toxins  do  not  deteriorate  or  vary  as  do  toxins) . 
A  standard  anti-toxin  made  by  Ehrlich  is  now  everywhere  used,  and 
is  furnished  by  him  from  his  institute. 

Against  this  standard  anti-toxin  a  toxin  of  unknown  strength   is 
measured  by  means  of  guinea  pigs.     The  toxin  unit  thus  founH  is 
then  used  to  determine  the  anti-toxic  unit  of  anti-toxins  of  unknown 
power. 
5 


66  IMMUNITY 

The  power  of  anti- toxic  sera  varies;  some  contain  from  200  to  300 
units  only  per  c.c;  others  may  contain  even  1,700  or  2,000  per  c.c. 

Anti- toxic  serum  is  preserved  by  the  addition  of  .5  percent  of 
tri-cresol  or  phenol.  It  remains  practically  unchanged  in  strength 
for  a  year  or  more.  When  used  it  is  common  to  inject  from  2,000 
to  5,000  units. 

It  is  not  only  of  value  as  a  curative  agent,  neutralizing  the  toxins 
already  formed,  but  is  valuable  as  an  immunizing  one  against  infec- 
tion. If  injected  early  in  a  case  of  diphtheria,  it  is  much  more  likely 
to  do  good,  than  if  used  later.  Some  desperate  cases  have  received 
100,000  units  and  have  recovered. 

Tetanus  Anti-toxin. — Tetanus  anti-toxin  is  produced  in  a  man- 
ner similar  to  that  of  diphtheria  anti-toxin.  As  the  horse  is  exceed- 
ingly sensitive  to  tetanus  toxin,  before  the  immunizing  process  is 
begun,  the  toxin  is  attenuated  by  heat  or  iodine. 

The  anti-toxin  is  standardized,  as  in  diphtheria,  by  testing  its 
potency  against  the  toxin.  A  guinea  pig  of  500  grams  weight  is 
used,  and  test  toxin  is  employed  of  such  strength  that  .01  c.c.  will 
kill  this  guinea  pig  in  about  four  days.  This  amount  of  toxin  is 
neutralized  by  ^^-^Vo^  of  a  unit  of  anti-tpxin,  or  one  unit  of  anti- 
toxin will  protect  1,000  guinea  pigs  against  the  minimum  fatal  dose 
of  tetanus  toxin.  The  United  States  unit  of  tetanus  anti-toxin  is 
now  the  least  quantity  of  anti-tetanic  serum  necessary  to  save  the 
life  of  a  350  gram  guinea  pig  for  ninety-six  hours  against  the  official 
test  dose  of  standard  toxin  furnished  by  the  Hygienic  Laboratory 
of  the  Public  Health  Service. 

There  can  be  no  doubt  that  tetanus  anti-toxin,  if  given  with  the 
toxin  or  soon  afterwards,  is  a  potent  means  of  preventing  lethal 
action  of  the  toxin.  Tetanus  toxin  enters  into  such  quick  combina- 
tion with  the  cells  of  the  motor  elements  of  the  nervous  system,  and 
the  union  is  so  permanent  that  it  is  difficult  for  the  anti-toxin  to  form 
any  union  with  the  combined  toxin.  If  an  immune  animal  whose 
blood  is  powerfully  anti-toxic  received  into  his  central  nervous  sys- 
tem a  dose  of  toxin  he  will  succumb  at  once;  the  anti- toxin  appar- 


ANTI-TOXINS  67 

ently  has  an  inferior  valency,  or  combining  power.  If  however,  it 
meets  the  toxin  before  it  reaches  the  nervous  system,  it,  by  its 
receptors,  binds  the  haptophores,  and  this  prevents  any  combina- 
tion of  the  toxophores  with  the  receptors  of  the  nervous  system  cells. 

In  general,  anti-toxin  is  effectual  if  administered  when  acute 
toxic  manifestations  of  the  disease  are  in  evidence.  It  has  been 
found  by  Calmette  and  McFarland  that  if  dried  tetanus  anti-toxin 
is  sprinkled  over  wounds  infected  with  tetanus  bacilli,  or  impreg- 
nated with  toxin,  that  it  acts  in  a  very  prompt  and  effectual  and  anti- 
dotal way. 

If  the  toxic  symptoms  appear  shortly  after  the  infecting  wound  is 
received  it  is  well  known  that  the  prognosis  is  extremely  grave.  In 
such  cases,  and  in  those  that  come  to  the  care  of  the  physician  late 
and  after  the  toxic  symptoms  have  appeared,  the  anti-toxin  must 
be  used  in  large  amounts  directly  about  the  wound  to  neutralize 
the  uncombined  toxin,  into  the  general  circulation  and  directly  into 
the  nervous  tissues  or  into  the  ventricle  of  the  brain,  in  the  hope  that 
the  excess  of  free  receptors  of  the  anti- toxin  may  engage  the  hapto- 
phores and  toxophores  of  the  toxin  molecule  already  attached  to 
the  receptors  of  the  nervous  cells  in  the  floor  of  the  fourth  ventricle. 

Streptococcus  Anti-toxin. — While  there  are  at  least  several 
strains  of  streptococci,  it  is  a  fact  that  the  toxins  produced  by  all 
have  the  same  charactertistics  and  properties.  The  toxin  is  of 
the  nature  of  a  diastase,  which  is  destroyed  by  a  temperature  of 
70°  C.  In  addition  to  the  ferment  of  a  diastatic  nature  others  of 
haemolytic  power  are  formed.  This  is  called  streptococcolysin  and 
it  is  said  by  Reudiger  to  possess  both  haptophorous  and  toxophor- 
ous  chains  in  the  toxin  molecule.  It  is  also  destroyed  at  70°  C. 
Jaundice  and  petechial  rash  is  often  found  in  streptococcic  infec- 
tions. It  causes  a  blood  stained  oedema  and  exudate  at  the  site  of 
infection  in  rabbits  killed  by  the  injection. 

The  anti-toxin  is  prepared  by  injecting  horses  with  living  cultures 
of  very  virulent  streptococci,  beginning  with  small  doses  and  increas- 
ing them  gradually.     The  last  dose  administered  in  the  immunizing 


68  IMMUNITY 

process  may  be  600  c.c.  of  a  virulent  culture.  Four  weeks  after  the 
last  dose  is  given  the  serum  is  withdrawn.  It  is  thought  by  Mar- 
morek  that  the  action  of  the  serum  is  anti-bacterial,  rather  than  anti- 
toxic. It  has  been  found  that  the  use  of  streptococci  from  human 
sources  is  the  most  efficient  for  the  immunization  of  horses. 

Anti-streptococcus  serum  is  of  some  value  in  infection  and 
diseases  caused  by  streptococci.  Of  these  it  has  been  used  in  puer- 
peral fever,  erysipelas,  and  septicaemia.  It  has  by  no  means  won  an 
undisputed  place,  like  diphtheria  anti-toxin. 

The  Anti-pneumococcus  serum  is  prepared  in  the  same  way. 
Horses  are  immunized  by  the  injection  of  living  cultures,  and  the 
horse's  blood,  after  a  period  of  treatment  by  cultures,  is  drawn  off, 
preserved  with  tri-cresol  and  used  in  a  manner  similar  to  diphtheria 
anti-toxin.  Its  use  has  not  been  attended  with  any  marked  results. 
It  is  a  curious  phenomenon  that  pneumococci  grow  better  in  the 
serum  of  a  horse  immunized  against  pneumococci  than  in  noimal 
horse  serum.  Autolysates  of  virulent  pneumococci  are  now  used 
for  immunizing  animals.  These  seem  to  raise  the  anti-bodies 
better  than  whole  cocci. 

There  are  anti-toxic  sera  for  use  against  Botulism,  or  meat  pois- 
oning, pyocyaneus  infection,  hay-fever,  staphylococcus  infec- 
tion, Malta  fever,  and  typhoid,  that  have  been  used  without  much 
success.  They  have  a  certain  scientific  interest,  but  are  of  no  great 
clinical  value. 

Anti-plague  Serum. — Yersin,  a  French  bacteriologist,  treated 
horses  with  living  cultures  of  plague  bacilli,  and  after  a  long  period 
of  immunization  used  a  serum  which  either  effectually  vaccinated  an 
individual  against  the  plague,  or  greatly  modified  the  disease  after  it 
had  once  begun.  Later  it  was  found  that  heat  killed  cultures  were 
just  as  effectual. 

In  142  cases  of  plague  treated  with  serum,  24  died,  a  mortality 
of  14.78  percent,  while  in  72  cases  untreated,  46  died,  a  mortality 
rate  of  63.72  percent. 

The  action  of  the  serum  is  bactericidal,  as  well  as  anti-toxic.  The 


VACCINATION  AGAINST  SMALL-POX  69 

dose  varies  with  the  stage  of  the  disease;  5  c.c.  is  an  effective  pro- 
phylactic dose,  while  from  20  to  300  c.c.  have  been  used  often  as 
curative  doses. 


VACCINATION. 

By  the  use  of  attenuated,  or  killed  micro-organisms,  it  is  possible 
to  effectively  vaccinate  men  and  animals  against  many  diseases, 
notably,  small-pox,  hydrophobia,  plague,  cholera,  typhoid  fever, 
anthrax  and  quarter-evil. 

Any  of  the  bacterial  products  used  as  prophylactics  are  sometimes 
called  vaccines,  the  word  being  borrowed  from  small-pox  vaccine. 
It  is  better  to  use  the  word  bacterin  for  the  purpose,  even  when  they 
are  given  •  prophylactically.  Bacterin  is  employed  for  the  dead 
bacterial  masses  used  therapeutically. 

Vaccination  Against  Small-pox. 

There  is  now  no  doubt  that  vaccinia  or  cow-pox  is  but  modified 
small-pox  in  the  cow.  The  causal  agent  of  small-pox,  through  its 
life  in  the  tissues  of  the  cow,  becomes  so  modified  that  it  does  not 
produce  in  man  variola,  but  vaccinia.  This  causal  agent  is  believed 
to  be  a  protozoan,  called  by  its  discoverers  Cytoryctes  variolce. 

By  the  term  vaccination,  in  its  strict  sense,  we  mean  the  applica- 
tion of  attenuated  small-pox  virus,  weakened  by  passage  through 
kine,  to  human  beings  and  infecting  them  with  the  modified  disease. 
The  disease  is  localized  at  first  at  the  site  of  inoculation,  and  a  bleb 
or  vesicle  forms.  As  a  rule  the  disease  does  not  become  generalized. 
It  creates,  in  the  vaccinated  individual,  an  active  immunity  against 
small-pox.  The  toxins  diffused  through  the  blood-stream  stimulate 
the  cells  of  the  body  into  forming  either  anti-toxic  bodies,  or  anti- 
body substances. 

These  various  substances,  as  yet  unknown,  remain  for  along 
period  within  the  body  of  the  vaccinated  person  and  may  protect  it 


70  IMMUNITY 

• 

for  years  against  invasion  and  infection  with  the  cytorcytes  in  viru- 
lent form.  A  person  who  has  variola  cannot  be  vaccinated,  sub- 
sequently he  is  immunized  against  vaccinia  by  this  attack  of  variola, 
just  as  he  can  be  immunized  against  variola  by  vaccinia  infection. 

Since  Jenner  first  discovered  that  cow-pox  introduced  into  the 
body  prevented  small-pox,  it  has  been  the  world-wide  custom  to  use 
either  the  dried  virus  or  liquid  glycerinized  virus  from  the  cow  or 
human  beings  in  the  process  of  vaccination.  It  has  been  found  that 
human  virus  generally  used  was  likely  in  rare  instances  to  transmit 
syphilis,  so  it  is  now  the  universal  custom  to  use  cow  virus.  This 
virus  is  collected  from  fresh  vesicles  in  calves  or  young  heifers,  as 
clean  as  possible,  as  it  is  used  as  seed  to  inoculate  the  animals  and 
the  operation  is  done  under  strict  anti-septic  precaution.  After  a 
week  the  virus  is  collected  under  similar  anti-septic  precautions  by 
scraping  the  base  of  the  vesicle  with  a  sterile  curette.  The  pulpy 
substance  thus  obtained  is  mixed  with  glycerine  and  stored  for  a 
month  or  more.  The  action  of  the  glycerine  is  to  rid  the  virus  of 
many  of  the  bacteria,  through,  it  is  supposed,  a  hydrolytic  action. 
This  virus  is  then  rubbed  into  the  skin  of  the  individual  to  be  vacci- 
nated under  strict  aseptic  precautions.  At  the  end  of  a  week,  a 
pearly  white  vesicle  is  formed,  and  it  is  then  considered  that  vacci- 
nation has  "taken"  and  that  the  individual  is  protected  against 
variola.  This  action  of  immunization  is  supposed  to  be  complete 
on  the  fourth  day  after  the  virus  has  been  introduced.  This  is  a 
matter  that  is  difficult  to  decide,  but  the  immunization  process  is,  no 
doubt,  a  very  slow  one,  like  every  other  immunizing  process  where 
the  immunity  is  autogenous  and  active,  and  not  passive,  as  in  the 
case  of  diphtheria  anti-toxin. 

There  can  be  no  doubt  about  this  being  one  of  the  greatest  boons 
that  mankind  has  ever  received.  Vaccination  is  attended  with  some 
risk.  Septic  infection  with  streptococci  sometimes  follows,  likewise 
tetanus  infection.  In  both  instances  this  may  be  due  to  the  contami- 
nation of  the  vesicle  on  the  calf  before  the  virus  is  lifted,  to  pirty 
methods,  or  to  contamination  after  vaccinations,  probably  the  latter. 


VACCINATION  AGAINST  CHOLERA  71 

Vaccination  Against  Cholera. 

By  the  injection  of  the  bodies  of  dead  bacteria,  or  attenuated  Hve 
ones,  especially  those  containing  in  their  cells  insoluble  poisons,  it 
is  possible  to  create  in  the  animals  experimented  upon  a  powerful 
active  immunity  against  the  action  of  living  virulent  bacteria  of  the 
same  species. 

By  the  attenuations  of  cholera  spirilla,  Haffkine  has  produced  vac- 
cines vi^hich  effectively  protect  individuals  against  infection  with 
cholera,  or  if  they  become  infected  vdth  the  disease,  it  is  so  modified 
that  they  can,  and  do,  more  easily  recover.  He  employs  two  vac- 
cines, a  weak  one  and  a  stronger  one.  The  weak  one  is  used  to 
prepare  for  the  stronger  one,  which  is  the  effective  vaccine. 

The  weak,  or  first  virus,  is  prepared  by  growing  the  cholera  vibrios 
at  a  high  temperature,  39°  C,  in  a  current  of  air.  The  stronger  is 
prepared  by  passing  the  vibrios  through  a  series  of  guinea  pigs,  so 
increasing  the  virulence  that  the  virus  is  invariably  fatal  to  the 
guinea  pigs  in  eight  hours.  The  best  method  is  to  use  a  culture  that 
kills  a  guinea  pig  in  twenty-four  hours  by  peritoneal  injection.  After 
the  am'mal  is  dead,  the  peritoneal  exudate  is  collected,  and  grown  at 
35°  C,  the  most  favorable  temperature  for  the  organism  to  multiply. 
This  exudate  is  injected  into  a  second  guinea  pig,  and  its  exudate  is, 
after  incubation,  injected  into  guinea  pig  number  three,  and  the 
process  is  done  repeatedly  until  the  virulent  virus  that  is  lethal  in 
eight  hours  is  obtained.  This  is  called  virus  fixe.  After  cultivating 
this  virus  on  agar,  the  surface  growth  is  washed  off  with  sterile  water 
(8  c.c.)  and  J  part  of  this  is  used  as  a  dose.  As  the  virus  rapidly 
attenuates  it  must  be  reactivated  by  passing  it  through  guinea  pigs 
from  time  to  time. 

The  first  injection  is  given  in  the  flank,  and  the  second  follows 
in  five  days.  Accordingly  as  the  symptoms  are  severe,  so  will  the 
resulting  protection  be  strong.  Haffkine  has  given  70,000  injections 
without  an  accident.  .  The  following  results  were  obtained  by  Haff- 
kine who  worked  in  India  for  the  British  Government: 


72 


IMMUNITY 


Population 

Cases— Cholera 

Deaths 

Total 

Percent 

Total 

Percent 

Non-inoculated,  1,735 

Inoculated,  500 

171 
21 

10.63 

4.2 

19 

6.51 
3.3 

The  immunity  conferred  by  this  mode  of  vaccination  is  not  com- 
plete until  ten  days  after  treatment.  It  is  possible  to  vaccinate  with 
these  relatively  virulent  bacteria  because  they  are  given  under 
the  skin,  a  place  where  the  life  of  the  vibrios  soon  ceases.  During 
an  attack  of  cholera  the  vibrios  do  not  enter  the  blood  but  remain 
in  the  deep  layers  of  the  intestinal  mucosa. 


Vaccination  Against  Typhoid. 

By  the  injection  of  sterilized  cultures  of  typhoid  bacilli,  it  is  pos- 
sible to  create  an  immunity  of  a  moderate  kind  against  enteric  fever. 
The  method  has  been  perfected  by  Wright,  and  his  mode  of  proce- 
dure is  to  secure  a  virulent  culture  of  typhoid,  which  is  tested  on 
guinea  pigs,  and  the  minimum  lethal  dose  for  a  100  gram  guinea  pig 
is  used  as  the  dose  for  man.  This  dose  varies  from  .5  c.c.  to  1.5  c.c 
of  an  old  culture  sterilized  by  heat  at  60°  C,  and  preserved  with 
lysol.  After  the  injection  there  is  often  redness  and  pain  at  the  site 
of  inoculation,  some  fever  and  lymphangitis.  The  results  obtained 
in  vaccinating  the  troops  in  South  Africa  are  marked.  Of  the  garri- 
son of  Ladysmith  comprising  nearly  12,000  troops,  1,705  were  inoc- 
ulated; 2  percent  contracted  typhoid  afterward,  and  4  percent  of 
these  died  of  the  disease.  Among  the  non-inoculated,  numbering 
10,529,  14  percent  contracted  typhoid,  and  3.12  percent  of  10,529 
died  of  the  disease.  It  seems  that  this  form  of  vaccination,  in  a 
great  measure,  prevents  the  infection  with  typhoid,  and  modifies  the 
disease  after  infection  occurs. 


ANTI-TYPHOID  VACCINATION  73 

The  results  of  Major  F.  F.  Russell,  U.  S.  A.,  a  man  who  has  had 
much  experience,  since  he  was  in  charge  of  the  army  vaccinations, 
are  interesting  and  instructive.     He  says: 

1.  "Anti-typhoid  vaccinations  in  healthy  persons  is  a  harmless 
procedure. 

2.  It  confers  almost  absolute  immunity  against  infection. 

3.  It  is  the  principal  cause  of  the  immunity  of  our  troops  against 
typhoid  in  the  recent  Texas  maneuvers. 

4.  The  duration  of  the  immunity  is  not  yet  determined,  but  is 
assuredly  two  and  one-half  years  and  probably  longer. 

5.  Only  in  exceptional  cases  does  its  administration  cause  an 
appreciable  degree  of  personal  discomfort. 

6.  It  apparently  protects  against  the  chronic  bacillus  carriers, 
and  is  at  present  the  only  means  by  which  a  person  can  be  protected 
against  typhoid  under  all  conditions. 

7.  All  persons  whose  profession  or  duty  involves  contact  with  the 
sick  should  be  immunized. 

8.  The  general  vaccination  of  an  entire  community  is  feasible 
and  could  be  done  without  interfering  with  general  sanitary 
improvements  and  should  be  urged  wherever  the  typhoid  rate  is 
high." 

The  present  method  is  to  give  three  injections  six  to  ten  days 
apart  of  definite  numbers  of  typhoid  bacilli  of  a  strain  known  to 
produce  a  good  quantity  of  agglutinins  and  other  anti-bodies.  The 
injections  usually  number  100,000,000,  500,000,000  and  1,000,- 
000,000. 

Vaccination  Against  Plague. 

Haffkine,  in  India,  has  vaccinated  many  natives  and  others 
against  plague  by  somewhat  the  same  methods  employed  in  anti- 
cholera  vaccination.  The  B.  pestis  is  cultivated  in  flasks  of  bouillon ; 
as  it  grows,  the  stalactite-like  scum  on  top  is  shaken  from  time 
to  time  to  the  bottom  of  the  flask.     After  growing  for  six  weeks  in 


74  IMMUNITY 

the  bouillon,  the  culture  is  killed  at  70°  C.  for  three  hours.  It  is 
then  used  as  vaccine,  3  c.c.  is  the  usual  dose  for  man,  2  c.c.  for 
woman,  and  children  still  less.  After  the  inoculation,  heat  and 
redness  appear  at  the  site  of  inoculation,  and  the  patient  feels  ill 
and  has  some  fever.  Haffkine  holds  that  immunity  against  the 
plague  is  complete  in  twenty-four  hours  after  vaccination.  His 
results  are  at  times  really  very  good.  In  a  village,  Unhera,  among 
64  uninoculated  people,  there  were  2  7  cases  with  2  6  deaths.  Among 
71  inoculated  persons  under  the  same  conditions,  and  of  the  same 
families  as  the  uninoculated,  there  were  8  cases  and  3  deaths.  The 
fatalities  among  the  unvaccinated  exceeded  those  among  the 
inoculated  by  89.65  percent. 

The  Indian  Plague  Commission  reported  that  the  measure  was 
valuable  as  a  means  of  preventing  infection;  while  it  was  not  an  abso- 
lutely certain  means,  yet  it  sensibly  diminished  the  death  rate.  The 
immunity  lasts  about  a  month.  Such  vaccines  are  not  to  be  used 
after  attack  has  started.  Yersin  and  Wyssokowitsch  have  devised 
an  anti-toxic  and  bactericidal  serum  from  injecting  horses  and 
monkeys.     This  may  be  used  as  a  remedy. 

Vaccination  Against  Anthrax. 

Of  all  forms  of  vaccination  against  disease  with  attenuated 
bacteria  this  is  the  most  successful.  Its  use  is  confined  to  domestic 
animals,  sheep,  cattle,  and  horses,  and  has  reduced  the  mortality 
in  the  country  where  it  is  used  from  10  percent  to  .5  percent. 
The  method  requires  the  employment  of  two  vaccines  made  of 
attenuated  anthrax  bacilli.  No.  i  is  a  culture  of  bacilli  attenuated 
by  growing  them  at  a  high  temperature,  42.5°  C,  in  a  current  of 
air  for  twenty-four  days.  No.  2  is  grown  at  the  same  temperature 
for  only  twelve  days.  The  first  vaccine  is  used  to  immunize  the 
animal  against  the  second,  which  causes  a  marked  local  reaction, 
and  which  is  the  real  immunization  agent  against  infection  with 
virulent  anthrax  bacilli.  The  injections  are  given  about  one  week 
apart.     Many  State  Governments  as  well  as  the  Federal  Govern- 


VACCINATION  AGAINST  TUBERCULOSIS  75 

ment  of  the  Unites  States  supply  the  vaccine  gratis  to  stock  raisers 
and  others. 

A  valuable  anti  bacterial  serum  is  used  also  as  a  therapeutic 
measure-anthrax  infection. 

Vaccination  Against  Black-leg  or  Quarter-evil. 

Quarter-evil,  or  Rauschbrand,  is  due  to  a  specific  bacillus.  Vac- 
cination against  this  disease  may  be  accomplished  by  inoculating 
with  a  powder  consisting  of  dried  muscle  from  the  affected  part  of 
infected  animal.  There  are  two  vaccines,  No  i,  and  No  2.  The 
first  is  prepared  by  heating  (and  thus  attenuating)  the  bacilli  up  to 
103°  C.  The  second  is  prepared  by  raising  the  temperature  up  to 
93°  C.  These  vaccines  are  given  at  a  short  time  apart,  and  the 
immunity  is  effective.     The  method  is  valuable  to  stockmen. 

Vaccination  Against  Tuberculosis. 

It  is  possible  to  vaccinate  animals  against  tuberculosis  by  the 
use  of  attenuated  tubercle  bacilli.  To  accomplish  this,  the  sole 
requisite  is  to  so  weaken  the  bacilli  used  to  immunize,  that  there  is 
not  any  likelihood  of  causing  any  lesion.  By  long  cultivation  on 
culture  media  bacilli  are  so  attenuated  that  they  cannot  cause  harm 
to  a  guinea  pig,  even  if  repeatedly  injected.  Guinea  pigs  may, 
when  properly  treated,  live  long  after  inoculation  with  virulent 
bovine  bacilli,  but  at  no  time  do  they  become  wholly  immune; 
cows  may  be  immunized  against  bovine  bacilli  by  inoculating  them 
with  weak  human  cultures.  Koch's  new  tuberculin,  made  by 
grinding  to  a  powder  the  dried  bodies  of  tubercle  bacilli,  is  also  able 
to  set  up  an  immunity  in  animals,  and  to  a  limited  extent  in  man. 
It  is  used  in  several  large  sanitariums  devoted  to  the  cure  of  tuber- 
culosis, as  a  therapeutic  agent.  Those  using  it  claim  that  it 
immunizes  the  individual  and  thus  increases  his  resisting  powers. 
Webb  of  Colorado  claims  to  have  produced  immunity  in  monkeys 
and  children  by  injecting  exceedingly  small  numbers  of  living 
bacilli,  I,  2,  4,  8,  12,  18,  25,  etc. 


76  IMMUNITY 

The  Tuberculins. 

The  toxin  of  the  tubercle  bacilli  -(old  tuberculin)  is  prepared  by 
growing  the  organism  for  a  long  period  in  glycerinized  veal  broth, 
after  which  the  flasks  are  steamed  in  a  sterilizer  for  an  hour  or  more, 
and  then  the  bacilli  are  filtered  out  through  porcelain  filters.  The 
filtrate  is  reduced  by  boiling  to  -^  of  its  bulk,  and  to  this  a  half  of  one 
percent  of  carbolic  acid  is  added  as  a  preservative.  If  this  toxin, 
even  in  minute  doses,  is  injected  under  the  skin  of  a  tuberculous 
animal,  it  acts  as  a  powerful  poison.  In  a  few  hours,  it  causes  a 
rapid  rise  of  body  temperature,  accompanied  by  nausea  and,  per- 
haps, vomiting.  About  the  localized  foci  of  tuberculosis,  a  vigorous 
reaction  occurs.  Around  indolent  old  sores  and  other  lesions  there 
is  a  tendency  to  heal  by  the  casting  off  of  necrosed  tissues,  and  the 
infiltration  of  the  peritubercular  area  with  leucocytes.  In  lupus 
(tuberculosis  of  the  skin)  redness  and  heat  occur  about  the  lesion. 
This  febrile  phenomenon  following  the  injection  of  tuberculin  into 
tuberculous  animals  is  a  valuable  diagnostic  feature  toward  the 
recognition  of  tuberculosis  in  animals  and  in  man.  In  90  percent 
of  cases  the  reaction  is  trustworthy. 

Tuberculin  acts  as  a  fever  producer  in  an  unknown  way.  It  is 
supposed,  however,  that  the  intense  local  reaction  produces  fever 
through  active  tissue  changes. 

Its  use  in  man  has  been  much  questioned,  as  it  is  thought  by  some 
to  disseminate  the  disease  from  original  and  confined  foci.  This 
however  has  been  denied.  Many  able  clinicians  use  it  and  recom- 
mend it.     (Osier,  Trudeau,  Musser.) 

Koch's  new,  or  T.R.  tuberculin  was,  like  the  old,  designed  by  him 
as  a  therapeutic  agent  for  the  cure  of  tuberculosis.  It  is  made  by 
pulverizing  the  bodies  of  living  tubercle  bacilli  and  dissolving  the 
residuum  in  an  indifferent  fluid,  centrifuging  this  and  collecting 
the  sediment  which  is  Tuberculin  Rest,  T.R.  The  solution  above 
this  sediment  containing  soluble  substances  from  the  bacillary 
bodies  is   Tuberculin  Obers,  T.O.     It  produces  a  more  intense 


IMMUNIZATION  AGAINST  HYDROPHOBIA  77 

reaction  than  the  old  tuberculin.  Like  the  old,  it  is  used  in  the 
treatment  of  lung,  bone,  laryngeal,  and  skin  tuberculosis.  It 
certainly  causes  a  local  reaction  about  tubercular  foci,  and  no  doubt 
aids  in  the  formation  of  an  active  immunity  to  the  disease. 

The  dose  of  tuberculin  for  testing  purposes  varies  from  ^^  of  a 
milligram  to  5  mgs.  and  in  case  the  first  dose  does  not  produce  a 
reaction,  it  should  be  repeated.  For  therapeutic  purposes  one 
begins  with  an  injection  of  .0000001  grm.  or  smaller  and  increases 
slowly  according  to  the  patient's  condition.  TubercuUn  should 
only  be  administered  by  experts. 

Mallein. 

Mallein  is  a  preparation  made  from  the  toxin  of  the  glanders 
bacilli,  and  is  prepared  precisely  as  the  old  tuberculin.  By  increas- 
ing the  virulence  of  the  glanders  bacilli,  by  passage  through  a  series 
of  guinea  pigs,  a  highly  virulent  bacillus  is  obtained.  It  is  then 
grown  in  glycerinized  bouillon  for  a  month  at  37°  C.  The  resulting 
fluid  is  sterilized  by  heat  and  filtered  through  a  Pasteur  filter.  The 
filtrate  is  evaporated  to  half  its  quantity,  and  to  this  a  small  amount 
of  carbolic  acid  is  added  in  order  to  preserve  it.  Of  the  mallein  thus 
prepared  i  c.c.  should  kill  a  rabbit  in  one  to  two  weeks. 

In  a  horse  with  glanders,  the  injection  of  mallein  is  followed  by  a 
large  painful  swelling  at  the  injection  site.  With  this  there  is  a  rise 
of  temperature,  which  is  the  diagnostic  reaction  that  indicates  infec- 
tion with  glanders.  In  this  respect  the  reaction  is  like  tuberculin. 
In  healthy  horses  no  rise  of  temperature  follows  the  injection,  and 
the  resulting  swelling  more  quickly  subsides.  Mallein  has  been 
used  as  a  prophylactic  agent  against  glanders  with  some  success. 

Immunization  Against  Hydrophobia. 

While  the  actual  causal  agent  of  hydrophobia  has  thus  far  eluded 
bacteriologists,  certain  well  marked  histologic  lesions  have  been 
discovered  in  the  ganglia  of  the  central  nervous  system,  and  in  the 


78  IMMUNITY 

medulla,  which  are  not  found  in  any  other  disease.  This  dispels  all 
doubt  as  to  the  fact  that  hydrophobia  is  a  real  clinical  entity. 

It  is  possible  to  immunize  animals  and  man  against  this  disease, 
by  the  use  of  attenuated  virus.  In  common  with  many  other 
viruses,  that  of  hydrophobia  can  be  weakened  through  the  action  of 
either  heat,  drying,  light,  or  chemicals.  Pasteur  found  that  by  dry- 
ing the  spinal  cords  of  rabid  animals  for  two  weeks,  they  become 
totally  avirulent.  If  the  cord  is  dried  but  three  or  four  days,  the 
virulence  is  but  slightly  modified.  Immunity  to  rabies  can  be  pro- 
duced by  injecting  minute  quantities  of  the  poison,  and  then 
gradually  increasing  the  dose  until  virulent  virus  can  be  employed. 

Modification  of  the  amount  of  poison  used  may  be  affected  by 
employing  equal  quantities  of  spinal  cords  from  rabid  animals  that 
have  dried  varying  lengths  of  time.  The  vaccine  consists  of  pieces 
of  cord,  I  cm.  in  length,  from  rabbits  that  have  been  killed  by  inocu- 
lation with  fixed  virus.  This  is  emulsified  with  sterile  salt  solution. 
Cord  that  has  dried  for  fourteen  days  is  first  injected,  after  which 
cords  that  have  dried  fewer  and  fewer  days,  until,  finally,  one  that 
has  dried  only  three  days  is  injected.     - 

In  cases  of  bites  by  rabid  dogs  on  the  face  or  head,  the  vaccination 
must  be  rapid,  so  two  injections  per  diem  are  given.  In  Berlin  the 
weakest  injection  used  (the  first)  is  made  from  a  cord  that  has  dried 
but  eight  days,  and  the  course  is  much  quicker.  The  effect  of  this 
mode  of  inoculation  is  to  produce  in  the  bitten  individual  a  very 
rapid  active  immunity,  quicker  in  its  action  than  the  infection.  The 
treatment  is  solely  prophylactic  and  in  no  way  curative.  If  symp- 
toms of  rabies  have  set  in,  the  treatment  is  of  no  avail.  In  rabies 
the  incubation  period  is  about  six  weeks,  so  that  there  is  plenty 
of  time  to  immunize  the  patient  by  injection  with  attenuated  virus. 

Since  the  immunizing  process  is  always  begun  after  the  bite  of  a 
rabid,  or  supposedly  rabid  dog,  it  differs  from  other  vaccinations, 
which  are  resorted  to  before  infection. 

Results  of  Treatment. — In  rabies  the  total  mortality  before  the 
introduction  of  vaccination  was  not  less  than  lo  percent.     Among 


COLEY'S  FLUID  79 

the  same  class  of  patients  in  the  Pasteur  institutes,  the  death  rate 
of  all  cases,  early  and  late,  has  been  reduced  to  a  fraction  of  i  per 
cent.  Those  cases  in  which  the  bites  are  on  the  head,  are  always 
more  serious,  and  the  mortality  is  higher.  Like  tetanus  the 
virus  travels,  it  is  supposed,  from  the  site  of  injury  to  the  central 
nervous  system  by  way  of  the  nerves.  If  the  bite  was  on  the  toe, 
it  would  take  longer  for  infection  to  reach  the  brain,  than  if  it  was 
on  the  upper  lip.  This  is  a  very  plausible  explanation  of  the  vary- 
ing incubation  periods  in  both  tetanus  and  hydrophobia. 

Coley's  Fluid  in  the  Treatment  of  Tumors. 

This  method  of  treatment  is  in  no  wise  a  prophylactic  one,  but 
strictly  a  curative  one.  It  consists  in  the  injection  of  the  toxins  of 
streptococci,  in  the  hope  that  they  will  cause  a  shrinking,  or  disap- 
pearance of  malignant  sarcomata.  An  attack  of  erysipelas  (it  has 
long  been  observed)  occurring  in  a  patient  with  some  malignant  dis- 
ease, has  the  effect  of  causing  a  disappearance,  or  retrogression,  of 
the  tumors.  Artificial  infection  with  streptococci  was  then  practiced 
with  the  idea  that  it  might  produce  the  same  effect.  But  this  was 
found  to  be  dangerous.  Coley  prepared  toxins  of  streptococci  by 
allowing  them  to  grow  with  the  B.  Prodigiosus.  The  mixture  after 
a  long  period  of  incubation  was  sterilized  by  heat,  and  the  fluid  thus 
obtained  was  injected  into  the  tissues.  Virulent  strains  of  strep- 
tococci are  used  and  the  dose  of  the  dead  culture  is  about  half  a  drop 
given  under  strict  anti-septic  precautions.  Out  of  200  cases  many 
were  cured.  In  35  cases  treated  by  other  surgeons  26  tumors  dis- 
appeared, and  14  of  these  cases  were  alive  from  two  to  four  years 
after.  The  best  results  are  obtained  in  spindle  cell  sarcoma, 
and  the  poorest  in  the  melanotic  variety.  The  method  by  no  means 
should  be  employed  where  the  tumor  can  be  removed  by  operation. 
It  cannot  supplant  the  knife,  and  only  in  inoperable  cases  or  as  a 
supplementary  treatment  where  other  forms  of  treatment  are 
employed,  should  it  be  used. 


8o  IMMUNITY 

Opsonins  and  Opsonic  Index. 

Peculiar  substances  in  blood  serum  have  been  called  by  Wright  and  Douglass 
opsonins  {Greek:  prepare  food  for).  If  fresh  blood  is  mixed  with  an  emulsion  of 
some  bacteria  and  then  incubated  for  half  an  hour,  it  will  then  be  found  that  many 
of  the  bacteria  are  within  the  polymorphonuclear  leucocytes.  If  the  serum  is 
washed  away  from  the  leucocytes  before  adding  bacteria,  none  of  the  latter  will 
be  found  within  the  leucocytes.  This  proves  that  the  serum  has  some  influence 
on  phagocytosis.  In  order  to  show  that  this  effect  is  on  the  bacteria  rather 
thein  on  the  leucocytes,  the  bacterial  suspension  may  be  treated  with  some  serum 
for  half  an  hour  and  then  washed  free  from  this  serum  by  means  of  a  salt 
solution  in  a  centrifuge,  and  then  mixed  with  some  serum-free  leucocytes;  then 
it  will  be  found  that  phagocytosis  occurs  as  before.  The  bacteria  have  been 
"sensitized."     According  to  Wright  this  action  is  comparable  to  cooking. 

Phagocytosis  then  depends  upon  the  action  of  some  serum  upon  bacteria, 
which  are  coped  with  in  the  body,  first  by  the  action  of  the  serum,  and  then 
by  the  leucocytes.  This  opsonic  substance,  like  the  amboceptors,  sometimes 
disappears  from  the  blood.     It  is  thermostabile. 

The  quantitative  action  of  phagocytosis  may  be  estimated  by  Lelshman's 
method.  He  mixed  blood  and  an  emulsion  of  bacteria  in  salt  solution  in  equal 
quantities,  and  allowed  them  to  stand  for  30  minutes  in  the  incubator.  After 
this  the  mixture  was  stained  and  the  average  number  of  bacteria  per  leucocyte 
was  obtained.     The  result  was  known  as  the  phagocytic  index. 

Wright  has  devised  the  following  technique.  Young  cultures,  a  few  hours 
old,  are  employed.  These  are  scraped  off  agar  tubes  and  mixed  with  salt  so- 
lution. After  this  has  sedimented,  the  supernatant  fluid  is  separated  from  the 
bacterial  masses  by  a  centrifuge;  is  pipetted  off,  and  preserved. 

Washed  leucocytes  are  obtained  by  collecting  2  c.c.  of  blood  in  30  c.c.  of  salt 
solution  containing  i  percent  citrate  of  soda  to  prevent  blood  coagulation. 
The  serum  and  citrate  of  soda  are  separated  from  corpuscles  by  washing  twice 
in  a  centrifuge.  The  upper  layer  of  the  sediment  is  rich  in  washed  leucocytes, 
and  is  used  in  the  experiments. 

To  obtain  the  opsonic  index,  blood  serum  from  various  cases  is  collected.  In 
the  case  of  staphylococcus  infection — say  furuncle — the  blood  serum  is  drawn 
from  the  patient  and,  with  equal  portions  of  an  emulsion  of  staphylococci 
(young  culture),  and  a  suspension  of  washed  corpuscles,  is  thoroughly  mixed 
in  a  pipette,  which  after  the  ends  are  sealed,  is  placed  in  an  incubator  for  15 
minutes.  A  drop  of  the  mixture  is  then  spread  upon  a  slide;  fixed,  and  stained 
with  Jenner's  stain.  The  number  of  staphylococci  in  50  polynuclear  leucocytes 
is  determined  and  divided  by  50  to  obtain  the  average. 

At  the  same  time  that  this  experiment  is  being  performed,  some  norma  1 


LOCAL  REACTIONS  8 1 

serum  should  be  used  in  another  experiment;  an  emulsion  of  staphylococci  and 
washed  leucocytes  being  used  as  above.  After  pursuing  the  same  steps  in  this 
experiment  as  in  the  first,  the  average  number  of  staphylococci  per  leucocyte 
is  determined. 

To  obtain  the  opsonic  index,  it  is  necessary  to  know  the  ratio  of  staphylococci 
in  the  leucocytes  treated  with  the  furuncular  serum,  and  in  the  normal.  If  the 
normal  serum  leucocytes  contained  lo  staphylococci,  and  the  furuncular  serum 
contained  15,  the  index  would  be  1.5. 

In  the  case  of  tubercle  bacilli,  the  latter  must  be  heated  to  100°  C.  to  kill 
them,  otherwise  they  will  be  agglutinated  by  the  serum,  and  a  homogeneous 
emulsion  will  not  be  obtained.  After  heating,  the  clumps  must  be  broken  up 
by  grinding  the  masses  in  an  agate  mortar,  adding  a  little  salt  solution  from 
time  to  time  until  the  mass  is  thoroughly  broken  up.  The  bacilli  must  then, 
after  phagocytosis,  be  stained  by  carbol  fuchsin  and  decolorized  with  acid  alcohol. 
If  the  leucocytes  are  left  too  long  in  contact  with  the  organisms  they  may  become 
so  engorged  as  to  prevent  counting,  the  number  increasing  from  5.7  percent 
after  five  minutes  to  28.5  percent  in  two  hours. 

Highly  immunized  anti-bacterial  serums  have  much  greater  opsonic  powers 
than  have  normal  ones,  anti-streptococcus  and  anti-pneumococcus  sera  being 
especially  powerful  toward  streptococci  and  pneumococci.  It  is  possible  to 
increase  the  opsonic  powers  of  the  blood  of  an  individual  suffering  from  an 
infection,  by  vaccinating  him  with*  killed  cultures  of  the  organism  with  which 
he  was  infected. 

Wright  has  treated  tubercular  and  septic  infections  in  this  way  with  excellent 
results,  the  opsonic  index  of  the  individual  being  very  markedly  raised.  Others 
have  not  had  such  convincing  results  with  the  opsonic  index. 

The  Local  Reactions  or  Tests. — We  have  learned  in  the  past  few  years 
that  the  skin  and  mucous  membranes  will  react  more  or  less  specifically 
to  the  bacterial  proteins.  It  is  a  form  of  allergic  (see  page  58).  There  have 
been  developed  local  tests  for  tuberculosis,  syphilis,  typhoid,  glanders  and  other 
diseases.  The  first  two  being  the  most  important,  are  considered  below. 
The  others  are  of  similar  nature. 

Tuberculosis. — If  tuberculin  of  any  form  be  rubbed  into  an  abraded  skin 
area  or  injected  between  the  layers  of  the  skin  a  red  maculopapule  or  even  vesicle 
upon  an  inflamed  base  will  appear  within  24  hours.  There  may  be  a  mild  general 
reaction  of  fever  and  malaise.  A  positive  reaction  to  such  an  installation  simply 
indicates  the  presence  of  a  tuberculous  lesion  and  that  an  anaphylactic  state 
of  the  skin  exists  but  does  not  show  whether  or  not  the  lesion  is  active.  For  this 
reason  it  is  only  of  value  in  children  since  three-fourths  of  adults  are  believed 
to  have  a  healed  lesion  within  them.     Not  only  upon  the  skin  but  upon  the 


82  IMMUNITY 

conjunctiva  can  this  reaction  be  obtained.     These  skin  tests  are  called  the  von 
Pirquet's  cutaneous  or  Moro's  percutaneous  tests. 

Syphilis. — The  poison  of  the  Treponema  pallidum  is  called  luetin.  It  is 
made  by  grinding  up  in  salt  solution  a  culture  of  the  germ,  heating  the  resulting 
mass  to  60  C.  for  an  hour  and  preserving  it  with  phenol.  If  this  be  instilled 
into  an  abraded  skin  area  a  maculopapule  or  nodular  eruption  occurs  in  a 
syphilitic.  This  positive  outcome,  hov^^ever,  only  appears  in  late  cases,  those 
of  tertiary  stages  and  in  treated  cases.  It  therefore  complements  the 
Wassermann  reaction,  being  positive  vi^here  this  is  apt  to  fail. 

Carriers. — After  recovery  from  certain  diseases,  notably  typhoid  fever,  diph- 
theria and  cholera,  convalescents  may  carry  in  themselves  fully  virulent  germs 
with  no  outward  evidences  thereof.  Such  persons  are  called  '* carriers''  and 
are  of  the  highest  importance  in  hygiene.  The  reasons  for  this  condition  are 
several.  These  germs  may  be  removed  from  the  bodily  defenses  or  the  body 
may  be  immune  to  them;  again  they  may  be  fixed  or  fast  strains.  Wherever 
they  are  they  may  escape  and  infect  another  person.  After  typhoid  fever 
bacilli  remain,  in  the  gall  passages  and  bladder;  after  cholera  in  the  deep 
mucous  membranes  and  after  diphtheria  the  crypts  of  the  tonsils  or  the  naso- 
pharynx may  hold  them.  Vaccination  or  operation  may  be  needed  to  remove 
them.  Persons  never  known  to  have  had  enteric  fever  have  been  known  to 
harbor  bacilli  in  their  gall  bladder.  One  typhoid  carrier,  "Typhoid  Mary"  a 
cook,  is  known  to  have  infected  26  persons. 


CHAPTER  V. 


STUDY  OF  BACTERIA. 


Bacteria  are  studied  in  the  following  various  ways: 

1.  Morphological  characteristics,  form,  size,  motility,  presence  of 
spores,  granules,  capsules,  and  flagella.  Reaction  of  protoplasm  to 
dyes  and  reagents. 

2.  Characteristics  of  growth  in  culture  media;  appearances  of 
culture;  chemical  activities;  production  of  acid,  gases,  toxins,  colors, 
etc. ;  reactions  to  heat,  disinfectants,  light,  etc. 

3.  Study  of  the  action  of  bacteria  on  the  tissues  of  man  and 
animals,  and  of  the  toxins  on  the  tissues  and  functions  of  the  various 
organisms. 

The  simplest  way  to  study  bacteria  is  to  make  a  hanging  drop  of  a 
fluid  containing  bacteria,  and  observing  the  organisms  under  a 
microscope.  To  do  this,  a  cover-slip  is  used  and  a  slide  with  a  con- 
cavity ground  in  it.  A  drop  of  bacteria  laden  fluid  is  placed  on 
the  cover-glass,  and  after  the  edges  have  been  smeared  with  vaseline, 
the  cover-slip  is  inverted  over  the  concavity  in  the  slide,  and  the 
bacteria  can  then  be  examined  with  either  the  dry  ^  inch,  or  the 
y^2  oil  immersion  objective.  If  the  preparation  is  kept  warm  for 
some  time,  various  vital  phenomena  may  be  noted.  Direct  division, 
sporulation,  motility,  agglutination,  and  bacteriolysis  can  be  studied 
by  this  means.  Instead  of  using  a  fluid,  a  block  of  nutrient  agar 
may  be  cemented  to  the  cover-glass;  after  the  bacteria  have  been 
planted  on  the  agar,  the  various  vital  phenomena  may  be  noted. 

All  minute  bodies,  whether  they  be  bacteria,  dust  particles  or 
granules  of  india  ink  in  suspension,  exhibit  a  trembling  vibrating 
motion  called  the  Brownian  motion.     Motile  bacteria  either  move  so 

83 


84  STUDY   OF   BACTERIA 

swiftly  that  the  eye  can  hardly  follow  them,  or  they  may  merely 
roll  or  waddle  across  the  field  slowly.  Direct  division,  if  proceeding 
under  the  best  conditions,  requires  but  15  to  40  minutes.  It  is  best 
observed  in  a  warm  stage  or  when  working  in  a  room  kept  at  a 
temperature  of  35°  C.  Sporulation  occurs  differently  in  different 
species.  In  some  it  will  be  found  soon  after  the  culture  has  been 
removed  from  the  incubator,  while  in  others  several  hours  are 
required.  Sporulation,  it  must  be  remembered,  is  a  resistant 
stage  when  unfavorable  conditions  are  met. 

The  Gruber-Widal  reaction  is  thus  studied.  A  drop  of  the 
serum  and  bouillon  culture,  mixed  in  proper  proportions,  is  dropped 
on  a  cover-slip,  which  is  then  placed,  drop  downwards,  over  the 
cavity  of  the  slide  {hanging  dropj  fig.  21).     (See  Agglutination.) 

Staining  bacteria  is  a  matter  that  is  easily  accomplished,  and 
very  many  staining  solutions  and  methods  have  been  invented  for 
this  purpose. 

The  simplest  procedure  is  to  take  a  drop  of  pus,  blood  or  culture, 
and  spread  it  upon  a  very  clean  slide  with  a  sterilized  platinum 


Fig.  21. — Hanging  drop,  over  hollow  ground  slide.     (Williams.) 

needle.  The  matter  must  be  spread  thinly  and  evenly.  After  the 
water  has  evaporated  and  the  preparation  has  become  dry  without 
the  use  of  heat,  it  must  be  fixed.  To  do  this  various  agents  are  used. 
The  object  of  the  fixing  is  to  coagulate  the  protoplasm  of  the  cells, 
and  to  fasten  all  the  smeared  matter  fast  to  the  glass,  so  that  the 
staining  fluid  and  water  will  not  wash  them  off.  This  is  accom- 
plished, in  the  case  of  a  slide,  by  holding  it  in  the  apex  of  a  bunsen 
flame  until  quite  warm  to  the  hand.  Great  care  must  be  used  not  to 
char  the  film.  Experience  is  needed  to  fix  slide  smears  correctly. 
The  beginner  would  do  well  to  use  cover-slips.  If  a  cover-slip  is 
used  it  must  be  passed  through  the  flame  three  times  rapidly.     After 


STAINING  BACTERIA  85 

fixing  and  thorough  cooling,  the  staining  fluid  is  poured  on,  and 
after  remaining  a  few  minutes  is  poured  off  and  the  slide  is  washed, 
dried  by  blotting  paper,  and  examined.  If  a  cover-slip  has  been 
used  a  drop  of  balsam  is  put  upon  a  clean  slide  and  the  cover, 
smeared  with  stained  bacteria,  is  inverted  on  the  balsam.  Upon 
the  stained  bacteria  themselves  (if  a  cover-glass  has  not  been  used) 
or  upon  the  cover-slip  a  drop  of  cedar  oil  may  be  placed,  and  the 
preparation  examined  with  a  one-twelfth  objective.  This  is  one  of 
the  simplest  staining  procedures  practised  in  bacteriology.  Other 
more  complicated  methods  will  now  be  described. 

Besides  heat,  absolute  alcohol,  methyl  alcohol,  or  formalin  may 
be  used  as  fixatives.  Some  stains  are  made  up  with  methyl  alcohol, 
and  instead  of  fixing  by  heat,  the  stain  is  merely  dropped  upon  the 
dried  film,  and  the  bacteria  are  fixed  and  stained  by  the  same 
solution  at  the  same  time,  water  being  added  for  differentiation  at 
the  end. 

Aniline  dyes  are  almost  entirely  used  as  stains  in  bacteriology  and 
these  are  divided  into  two  classes,  the  basic  and  acid  stains,  accord- 
ing as  their  staining  properties  depend  upon  the  basic,  or  acid  part  of 
the  molecule.  Basic  dyes  stain  nuclear  tissues  of  cells  and  bacteria. 
The  acid  are  used  as  contrast  stains  and  do  not  color  bacteria,  but 
tissues  in  which  they  may  be  imbedded. 

The  common  basic  stains  are  methyl  violet,  and  gentian  violet, 
methyl  green,  methyl  blue,  and  methylene  blue,  thionin  blue,  Bis- 
marck brown,  fuchsin,  and  saffranin.  These  are  used  for  staining 
different  bacteria  under  different  conditions.  The  most  useful  stain 
is  methylene  blue,  since  it  is  difficult  too  verstain  with  it,  and  it  is 
very  easily  applied.  It  has  been  found  that  certain  physical  and 
chemical  conditions  are  necessary  for  successful  staining  with  ani- 
line dyes.  Alcoholic  solution  of  dyes  entirely  devoid  of  water  do 
not  stain,  absolute  alcohol  does  not  decolorize  bacteria  after  stain- 
ing with  aniline  colors,  while  diluted  alcohol  decolorizes  readily. 
The  more  completely  a  dye  is  dissolved,  the  weaker  is  its  staining 
power.     A  dye  stuff  unites,  as  a  whole,  with  the  bacterial  plasma. 


86  STUDY   OF   BACTERIA 

forming,  as  it  were,  a  double  salt  between  the  two.  Certain  sub- 
stances, alkalies,  carbolic  acid,  iron  and  copper  sulphate,  tannic 
acid,  alum,  and  aniline  oil,  are  added  to  a  solution  of  aniline  dyes, 
and  they  act  as  mordants,  or  fixatives,  making  the  dye  bite  into  the 
protoplasm  of  the  bacterial  cells.  Spores,  capsules,  and  flagella, 
are  hard  to  stain,  and  special  heavily  mordanted  stains  are  used  to 
demonstrate  them.  Chemical  reaction  occurring  in  the  cell  proto- 
plasm is  of  great  value  in  differentiating  bacteria.  The  presence  of 
granules  in  bacterial  cells  is  often  only  shown  by  the  use  of  special 
stains,  which  deeply  color  them.  Bacteria  of  the  tubercle  group  are 
called  "acid  fast,"  because,  after  being  stained,  it  is  difficult  to 
decolorize  them  with  acid  solutions.  These  bacteria  are  hard  to 
stain  and  resist  decolorizing  agents  after  they  are  stained. 

1.  Loffler's  alkaline  methylene  blue  solution  consists  of 

Saturated  alcoholic  solution  of  methylene  blue 30  c.c. 

To  00^  solution  caustic  soda  solution  in  water 100  c.c. 

Mix. 

This  is  the  most  useful  of  all  the  staining  mixtures  employed. 

2.  ZeihPs  solution  carbol-fuchsin  consists  of 

Fuchsin i  gram. 

Carbolic  acid  crystals 5  grams. 

Dissolved  in  100  c.c.  of  water,  to  which  is  added  10  c.c.  of  absolute  alcohol. 

This  can  also  be  made  by  taking  a  5  percent  solution  of  carbolic 
acid  in  water  and  adding  sufficient  saturated  solution  of  fuchsin  in 
water  until  a  bronze  scum  persists  upon  the  top.  This  is  used  for 
staining  tubercle  bacilli  in  sputum  and  sections.  It  must  be  heated 
when  used  for  rapid  staining.  Tubercle  bacilli  can  be  stained  in 
cold  solution,  if  immersed  over  night  in  it. 

3.  Fuchsin  solution. 

Saturated  alcoholic  solution  of  basic  fuchsin i  c.c. 

Water 100  c.c. 


STAINS  87 

4.  Bismarck  brown  solution. 

Water 100  ex. 

Bismarck  brown  sufficient  to  saturate. 
Filter  and  use  as  conlrast  stain. 

5.  Weigert's  aniline  gentian  violet  stain. 

Gentian  violet i  gram. 

Dissolve  in  absolute  alcohol 15  c.c. 

Distilled  water 80  c.c. 

Then  add  to  this 

Aniline  oil 3  c.Cc 

Mix,  shake  and  filter. 
This  stain  can  also  be  prepared  by  taking  a 

Sat.  watery  solution  of  aniline  oil 100  c.c. 

Filter,  then  add 

Sat.  alcoholic  solution  gentian  violet 10  c.c. 

This  is  a  very  intense  bacterial  stain  used  for  demonstrating 
bacteria  by  the  Gram  method. 

Gram's  method  of  staining. 

A  cover-glass  is  spread  with  a  smear  of  bacteria,  or  pus  to  be 
examined.  After  air-drying  it,  and  fixing  it  in  the  flame,  the  aniline 
gentian  violet  is  poured  on,  allov^^ed  to  stand  for  three  minutes, 
then  poured  off  and  the  preparation  treated  with 

Iodine  crystals i  gram. 

Potassium  iodide 2  grams. 

Water 100  c.c. 

for  two  minutes.  This  renders  the  purplish  preparation  grayish  in 
appearance.  Alcohol  is  now  poured  upon  the  preparation  repeat- 
edly until  the  alcohol  does  not  dissolve  any  more  color.  A  contrast 
stain  of  Bismarck  brown  or  dilute  fuchsin  is  now  used.  If  the 
bacteria  on  examination  remain  a  dark  violet  blue  they  are  then 
said  to  stain  by  Gram's  method,  or  are  "Gram  positive."  If  they 
are  decolorized  they  take  the  contrast  stain  and  are  said  not  to  stain 
by  this  method,  and  are  "Gram  negative." 


88  STUDY   OF   BACTERIA 

Many  bacteria  stain  in  this  way,  and  many  do  not.  Important 
bacteria  often  may  be  differentiated  in  this  manner. 

Examples  of  Gram's  stain  are  as  follows: 

Gram  positive — Bact.  aerogenus  capsulatu^  BvcL  anthracis,  Bad. 
diphtherice,  B.  tetani,  Bact.  tuberculosis,  Streptococcus  pneumonicB, 
Staph,  pyogenes,  Strep,  pyogenes.  Gram  negative — B.  coli,  B. 
dysenteric^,  Bact.  injiuenzce,  Bact.  mallei,  Bact.  pesth,  B.  pyocyaneus, 
B.  typhosus,  Diplococcus  intracellularis  meningitidis,  Micr.  catarrh- 
alis,  Micr.  gonorrhoece,  Spirillum  cholercB. 

Thionin  Blue,  or  Carbol  Thionin. 

This  is  a  useful  stain,  prepared  thus: 

Thionin  blue i      gram. 

Carbolic  acid 2.5  gram. 

Water 100  c.c. 

Filter.     Good  for  staining  bacteria  in  tissues. 


Special  Stains. 

Wright's  Stain. — This  not  only  stains,  but  fixes.  It  has  a  wide 
range  of  usefulness  in  a  bacteriological  laboratory  for  the  staining 
of  blood,  pus,  malarial  parasites,  trypanosomes,  as  well  as  many 
bacteria,  and  is  prepared  as  follows: 

•5%  solution  of  sodium  bicarbonate 100  c.c. 

Methylene  blue i  gram. 

Mix  and  heat  in  sterilizer  one  hour  at  100°  C.  Cool,  filter,  then  mix  ^(,  per- 
cent yellowish  eosin  in  water  until  the  mixture  loses  its  blue  color  and  becomes 
purplish.  Of  the  eosin  solution  add  500  c.c.  to  each  100  c.c.  of  the  methylene 
blue  mixture.  Mix  and  collect  the  abundant  precipitate  which  immediately 
forms  on  a  filter.  Dry  this  and  dissolve  in  methyl  alcohol  in  the  proportion  of 
I  gram  of  powder  to  600  c.c.  of  the  alcohol.  This  is  the  staining  fluid.  Keep 
well  stoppered.     Fresh  alcohol  may  be  added  for  that  which  evaporates. 

This  complex  stain  represents  a  type  of  which  Jenner's,  Leish- 


SPECIAL  STAINS  89 

man's,  and  Romanowsky's  are  members.  To  use  this  stain,  a 
blood  or  pus  film  is  spread  and  air  dried.  The  stain  is  then  run 
on  the  slip,  or  slide,  for  one  minute.  After  this  time  slowly  drop 
distilled  water  in  quantity  similar  to  that  of  stain  used.  This  is 
when  the  true  staining  takes  place.  After  three  minutes  wash  in 
distilled  water,  dry  and  mount.  Nuclei,  malarial  parasites,  trypano- 
somes,  and  bacteria  are  stained  blue;  red  cells  are  stained  pinkish- 
orange;  while  the  granules  of  the  leucocytes  are  stained  pink,  lilac, 
or  blue,  depending  upon  their  character. 

Giemsa's  Stain. 

This  stain  is  used  for  demonstrating  the  newly  discovered  organ- 
ism of  syphilis — Treponema  Pallidum  {Spirochcete  Pallida)  and  is 
prepared  as  follows: 

Azur  II  Eosin 3  grams. 

Azur  II 8  grams. 

Glycerine  C.  P 250  c.c. 

Methyl  alcohol 250  c.c. 

1.  Air  dry  the  specimen. 

2.  Harden  and  fix  in  absolute  alcohol. 

3.  Dilute  stain  with  distilled  water,  using  one  drop  of  stain  to  each  cubic 
centimeter  of  water. 

4.  Cover  preparation  with  dilute  stain  15  minutes. 

5.  Wash  in  running  water. 

6.  Blot  and  mount. 

Capsule  Staining. 

Bacteria  are  often  covered  with  capsules  that  are  difficult  to  stain, 
and  special  methods  have  been  devised  to  demonstrate  them. 

Welch's  Method. 

I.  Cover-glass  preparations  are  made  in  the  usual  manner,  and  over  the  film 
after  fixing,  glacial  acetic  acid  is  poured. 


90  STUDY   OF    BACTERIA 

2.  Without  washing  oflf  the  acid,  aniline  water  gentian  violet  is  poured  on. 
Change  the  stain  four  or  five  times  to  remove  the  acid.  Stain  four  minutes. 
This  demonstrates  the  capsule  very  well. 

His's  Method. 


1.  Make  cover-glass  preparation  as  usual.    Fix  in  flame. 

2.  Stain  for  a  few  seconds  with  a  half  concentrated  water  solution  of  gentian 
violet. 

3.  Wash  in  weak  potassium  carbonate  solution  for  a  few  minutes. 

4.  Dry  and  mount. 

"B". 

1.  Dry  and  fix. 

2.  Heat  and  pour  on  the  following  stain: 

a.  Saturated  alcoholic  solution  of  gentian  violet 5  c.c. 

b.  Water 95  c.c. 

3.  Wash  in  a  20  percent  solution  cupric  sulphate. 

4.  Dry  and  mount. 

Spore  Staining. 

Spores  resist  stains,  and  when  stained  are  hard  to  decolorize. 

1.  Dry  and  fix  in  the  usual  way. 

2.  Flood  cover-glass  with  hot  carbol-fuchsin;  heat  until  it  steams;  repeat 
this  once  or  twice.     This  stains  bacteria  and  spores. 

3.  Wash  in  water. 

4.  Decolorize  with 

Alcohol 2  parts. 

I  %  acetic  acid i  part. 

5.  Wash. 

6.  Counterstain  with  methylene  blue. 

7.  Wash,  dry  and  mount. 

By  this  method,  which  is  a  simple  and  satisfactory  one,  the  spores 
are  stained  a  brilliant  red,  while  the  body  of  the  bacilli  are  stained 
blue. 


FLAGELLA  STAINING  9I 

Flagella  Staining. 

To  a  beginner  flagella  staining  is  difficult;  there  have  been  many- 
well  known  methods  devised.  The  simpler  are  as  effective  as  the 
more  complicated  but  do  not  always  make  as  pretty  preparations. 

Flagella,  being  processes  extending  from  the  capsule,  are,  like  the 
latter,  hard  to  demonstrate.  They  are  not  stained  by  the  common 
bacterial  stains.  In  general  a  powerful  stain  mixed  with  a  strong 
mordant  must  be  employed.  Some  methods  appear  to  be  not  so 
much  a  staining  method  in  the  ordinary  sense  but  either  a  precipi- 
tating of  the  stain  in  the  substance  of  the  flagella  or  else  a  decom- 
position of  silver  salts  in  the  flagella  substance.  To  stain  flagella, 
a  young  culture  grown  on  agar  must  be  employed;  glycerine  agar 
must  never  be  used.  A  mass  of  the  organism  is  gently  mixed  with 
a  drop  of  distilled  water  until  a  uniform  emulsion  is  made.  A  dozen 
cover-slips  carefully  washed  and  cleaned  by  alcohol  are  thoroughly 
flamed  in  order  to  remove  the  slightest  trace  of  grease.  The  watery 
emulsion  of  bacteria  is  then  spread  over  the  cover-slips  evenly  and 
thinly.  After  they  are  dry  the  bacteria  are  fixed  by  holding  them 
for  a  minute  just  above  the  apex  of  the  flame  with  the  fingers.  The 
following  methods  may  be  pursued: 

Pitfield*s  Method  Modified  by  Muir. 

Two  solutions  are  necessary  for  this  method. 

A.  Mordant. 

10  percent  watery  solution  tannic  acid 10  c.c. 

Corrosive  sublimate  saturated  water  solution 5  c.c. 

Carbol-fuchsin  solution 5  c.c. 

This  forms  a  dense  precipitate  which  must  be  removed  by  the  centrifuge, 
or  sedimentation,  and  the  clear  fluid,  or  mordant,  is  stored  in  a  bottle.  It  keeps 
for  two  weeks. 

B.  Stain. 

Saturated  watery  solution  of  alum 10  c.c. 

Saturated  alcoholic  solution  gentian  violet 2  c.c. 

This  keeps  but  two  or  three  days. 


92  STUDY   OF    BACTERIA 

Flood  the  cover-slip  with  the  mordant  and  gently  steam  for  one 
minute,  then  wash  and  dry  thoroughly,  pour  the  stain  on  and 
steam  for  one  minute  more.     Wash,  dry  and  mount. 

This  method  yields  very  good  results. 

Pitfield's  Method. 

This  is  the  simplest  stain  and  the  easiest  to  use,  but  does  not 
give  the  good  results  that  the  previous  one  does.  But  one  solution 
is  needed,  this  is  made  in  two  parts  and  mixed. 

A.  Tannic  acid i  gram. 

Water lo  c.c. 

B.  Saturated  watery  solution  alum  (old) lo  c.c. 

Saturated  alcoholic  solution  gentian  violet i  c.c. 

Mix. 

A  heavy  precipitate  is  formed  by  this  process  v^hich  is  useful  in  the  stain- 
ing. The  stain  is  almost  a  saturated  solution  of  alum  and  tannic  acid,  and 
when  it  becomes  supersaturated  by  evaporation  and  heat,  staining  takes  place. 
After  this  the  process  is  very  simple.  The  cover-slip  is  carefully  flooded  with  the 
stain  and  warmed  for  a  minute  over  the  flame  of  a  bunsen  burner,  turned  very 
low,  until  steam  arises.  Not  too  much  stain  should  be  run  over  the  cover-slip. 
After  steaming  occurs,  the  stain  should  remain  for  a  minute,  then  the  preparation 
is  washed,  dried,  and  mounted.  It  will  be  found  that  the  best  stained  flageUa  are 
on  those  bacteria  nearest  to  the  edges  where  the  evaporation  has  been  most 
intense.  If  the  preparation  is  not  equally  stained,  Weigert's  aniline  gentian 
violet  can  be  run  on  for  a  minute  to  deepen  the  color. 

LoflBler's  Method. 

This  is  the  original  flagella  stain  and  is  a  very  good  one. 
It  is  made  as  follows: 

A.  Mordant 

20  percent  watery  solution  tannic  acid 10  c.c. 

Sat.  solution  ferrous  sulphate 5  c.c. 

Fuchsin  sat.  alcoholic  solution i  c.c. 

Mix 

B.  Stain 
Carbol-fuchsin. 

Proceed  as  in  the  previous  methods. 


STAINING  DIPHTHERIA  BACILLI  93 

The  most  important  steps  in  flagella  staining  are  to  clean  the 
cover-slips  thoroughly,  to  mix  the  culture  with  water  and  have  no 
culture  media  with  it,  to  fix  gently,  and  not  to  overheat  the  stain. 
Even  in  expert  practised  hands  it  is  not  always  easy  to  demonstrate 
flagella  readily. 


\ 


i 


Fig.  22 — B.  Diphtheria  stained  by  Neisser's  method.     (Williams.) 

Neisser*s  method  of  staining  the  diphtheria  bacillus. 
Two  stains  are  needed:   (Fig.  22.) 

A.  Methylene  blue i  gram. 

95  percent  alcohol 20  c.c. 

Water 950  c.c. 

Mix  and  add 

Glacial  acetic  acid 50  c.c. 

B.  Vesuvin 2  grams. 

Distilled  water 1000  c.c. 


The  staining  steps  are  as  follows: 

1.  Prepare  film,  fix  and  dry. 

2.  Pour  on  "A"  for  thirty  seconds. 


94  STUDY   OF   BACTERIA 

3.  Wash  well  in  water. 

4.  Dry  and  pour  on  "B"  for  thirty  seconds. 

5.  Wash,  dry  and  mount. 

The  protoplasm  of  the  bacilli  will  be  stained  brown,  and  the 
characteristic  (diagnostic)  chromatin  points  will  be  stained  a  deep 
blue  black. 


Tubercle  Bacillus  Stain. 

1.  Spread  the  sputum,  pus  or  culture,  over  the  surface  of  the  cover-slip. 
Allow  the  preparation  to  thoroughly  dry. 

2.  Fix  in  flame  and  cool. 

3.  Pour  carbol-fuchsin  over  the  slide  and  heat  with  steaming  for  five  minutes. 
Young  bacilli  in  tubercles  and  other  fluids  are  very  difficult  to  stain  in  this  way. 
The  preparation  containing  them  should  be  stood  in  cold  carbol-fuchsin  for 
twenty-four  hours.     This  method  stains  everything  on  the  slide. 

4.  Wash  in  water. 

5.  Decolorize  the  preparation  with  a  25  percent  solution  of  sulphuric  acid 
in  water  until  the  red  color  is  lost.     Repeat  this  once  or  twice. 

6.  Wash  and  counterstain  with  Loffler's  methylene  blue. 

7.  Dry  and  mount. 

In  such  a  preparation,  if  tubercle  or  other  acid-fast  bacilli  are 
present,  the  bacilli  will  be  colored  a  brilliant  red,  while  the  pus  cells, 
epithelial  cells,  and  other  bacteria  will  be  stained  blue. 

The  ultra  microscope  dark  field  illumination  enables  one  to  see 
fiagella  and  capsules.  This  illumination  is  obtained  by  blocking 
out  the  central  portion  of  the  Abbe  condenser  in  the  substage  of 
the  microscope.  Light  is  admitted  only  from  the  sides  and  objects 
in  the  field  at  the  point  of  crossing  of  the  rays  reflect  these  from 
their  sides.  India  ink  may  be  used  as  a  background  for  bacteria 
that  stain  poorly  and  have  low  refractive  index. 

Protozoa  are  stained  by  Wright's  method  in  one  of  its  various 
forms.  Microscopic  objects  are  measured  by  viewing  with  an 
ocular  fitted  with  a  graduated  glass  disc.  Their  values  are  indi- 
cated on  the  apparatus. 


DARK  BACKGROUND  ILLUMINATION  95 

Bacteria  may  be  most  beautifully  studied  by  means  of  the  dark 
field  method  of  illumination  in  which  they  appear  luminious 
against  a  black  background.  An  arc  light  and  an  especial  sub- 
stage  condenser  are  necessary.  By  mixing  a  bacterial  emulsion  in 
fluid,  such  as  blood  in  saliva,  with  a  mixture  of  India  ink  and 
water  and  drying  it  on  a  slide,  in  examination  the  bacteria  are  not 
stained  but  aie  sharply  defined  against  the  black  ink  in  a  beautiful 
way.  The  various  spirochaetas  and  trypanosomes  may  be  studied 
in  these  two  ways  very  satisfactorily. 


CHAPTER  VII. 

BACTERIOLOGICAL  LABORATORY  TECHNIC. 

In  order  to  study  bacteria  by  other  methods  than  the  simple 
examination  of  their  morphology  by  means  of  stains,  and  by  the 
hanging  drop,  or  block  method,  they  must  be  cultivated  either  in 
the  bodies  of  experiment  animals,  or  in  culture  media  artifici- 
ally prepared.  The  latter  method  is  the  most  widely  used  in 
laboratories.  It  is  necessary,  in  order  to  study  bacteria,  that  the 
media  shall  not  contain  any  extraneous  bacteria  to  begin  with, 
and  that  they  shall  be  cultivated  under  such  conditions  that  these 
bacteria  cannot  reach  the  media  at  any  time.  To  accomplish  all 
this,  the  culture  media  must  be  kept  in  glass  vessels,  such  as  test- 
tubes  and  flasks  that  have  been  sterilized.  And,  since  all  animal 
and  vegetable  substances,  not  actually  alive,  are  overwhelmed 
with  a  multitude  of  bacteria,  these  substances  must  be  sterilized 
too,  in  order  that  the  media  shall  be  free  from  any  living  organisms. 

Glassware,  such  as  pipettes,  Petri  dishes,  flasks  and  test-tubes, 
are  sterilized  best  by  dry  heat  in  hot  air  sterilizers.  The  apparatus 
is  subjected  to  a  temperature  of  150°  C.  for  one  hour,  or  until  the 
cotton  plugs  are  slightly  brown.  The  glassware  should  be  put  in 
wire  baskets  and  the  test-tubes  should  be  kept  erect.  Petri  dishes 
are  best  sterilized  in  a  wrapping  of  paper.  Flasks  and  test-tubes 
are  always  plugged  with  raw  cotton,  which  prevents  the  ingress  of 
bacteria,  while  air  can  reach  the  media  through  it  freely. 

Sterilization  of  culture  media  is  accomplished  in  steam  sterilizers 
of  two  patterns;  of  these,  the  autoclave,  using  steam  under  pressure, 
is  the  most  satisfactory  and  is  most  generally  used  at  present. 

The  baskets  containing  the  culture  media  are  placed  in  the  auto- 

96 


STERILIZATION 


97 


clave  after  two  quarts  of  water  have  been  put  in  it.  The  lid  is 
screwed  down  and  the  flame  started;  free  flowing  steam  should 
escape  from  the  valve  before  the  latter  is  shut.  When  the  pressure 
has  risen  to  one  atmosphere  (15  pounds)  or  120°  C.  for  twenty 
minutes,  all  bacteria  are  destroyed,  and  the  media  can  be  safely 
assumed  to  be  sterilized.     If  media  containing  sugar  or  gelatine  are 


Fig.  23. — Autoclave. 


to  be  sterilized,  the  temperature  should  not  run  above  110°  C,  since, 
if  this  is  done  the  gelatine  will  not  solidify  when  cold,  the  sugar  is 
caramelized  and  the  media  blackened. 

Potato  tubes  are  harder  to  sterilize  a  times,  and  it  is  safer  to 
repeat  the  operation  in  twenty-four  hours. 

Fractional  method  of  sterilization,  or  Tyndallization,  is  accom- 
7 


98 


BACTERIOLOGICAL   LABORATORY  TECHNIC 


plished  by  heating  the  media  to  ioo°  C.  on  three  successive  days 
in  a  Koch  or  Arnold  sterilizer.  By  heating  culture  media  to  this 
temperature,  all  the  vegetative,  or  adult,  forms  are  killed,  v^hile  the 
spores  are  not  affected;  after  the  first  sterilization,  at  room  tempera- 
ture, the  spores  vegetate  and  become  adult  bacteria,  when  on  the 
second  sterilization  they  are  non-resistant  to  ioo°  C.  and  are  killed. 


Fig.   24. — Arnold  sterilizer. 

Spores  remaining  after  this  develop  into  adult  forms  again 
and  are  killed  on  the  third  day,  at  the  third  sterilization.  This 
fractional  sterilization  is  employed  in  many  laboratories  still,  and 
is  certainly  the  best  for  media  containing  carbohydrates  of  any 
kind.  To  be  effective,  the  media  must  be  exposed  to  a  temperature 
of  100°  C.  for  thirty  minutes,  that  is,  thirty  minutes  after  the  steam 
has  begun  to  form.  Over  heating  of  sugars  causes  them  to 
caramelize  and  turn  black. 


BACTERIA  CULTIVATION 


99 


Bacteria  that  grow  best  at  a  temperature  of  37°  C.  (most  of  the 
pathogenic  ones  do)  develop  more  rapidly  and  luxuriantly  in  an 
incubator,  or  thermostat.  Indeed  some  organisms,  like  the  tubercle 
bacillus,  cannot  be  cultivated  without  it.  An  incubator  comprises, 
an  air  chamber  surrounded  by  a  water  chamber,  and  this,  in  turn, 
is  surrounded  by  another  air  chamber.  It  is  essential  that  the 
interior  of  the  incubator  be  kept  at  an  even,  unvarying  temperature. 


Fig.  25. — Incubator. 

This  is  accomplished  by  using  a  small  bunsen  flame  under  the  incu- 
bator. The  heat  from  the  flame  warms  the  outer  air  chamber  or 
jacket,  and  it  in  turn  warms  the  water  jacket,  and  the  interior  air 
chamber,  where  the  cultures  are  kept,  is  thus  heated  to  the  required 
temperature.  The  amount  of  heat  is  automatically  regulated  by  a 
thermo-regulator,  which  diminishes  the  gas  supply  if  the  temper- 
ature runs  too  high,  or  increases  it  if  it  runs  too  low.  The  Roux 
regulator  is  the  simplest  and  most  efficient  one. 


lOO  BACTERIOLOGICAL   LABORATORY   TECHNIC 

A  serum  coagulating  apparatus  is  needed  in  laboratories  in  order 
to  coagulate  the  tubes  of  blood  serum.     (Fig.  26.) 

Serum  tubes  are  coagulated  in  it  at  a  temperature  of  about  70°  C. 
They  are  then  sterilized  by  heating  them  for  an  hour  at  this  tem- 
perature, for  five  successive  days. 

The  separation  of  bacteria  from  the  bouillon  in  which  they  grow 


Fig.  26. — Blood  serum  coagulating  apparatus. 

for  the  preparation  of  toxins  requires  the  use  of  a  bacteria  or  germ 
proof  filter,  the  best  type  of  which  is  the  Chamberland  or  Pasteur 
unglazed  porcelain  filter.  These  filters  are  of  varying  grades  of 
fineness,  and  are  so  made  as  to  be  easily  sterilized.  The  common 
pathogenic  bacteria  cannot  pass  through  the  pores  of  the  ordinary 
filter,  but  toxic  agents  are  known  to  pass  through  the  finest  filters, 
though  they  cannot  be  discovered,  as  they  are  submicroscopic. 

To  operate  the  porcelain  filter  it  must  fit  into  the  nepk  of  a  vessel 
very  tightly,  so  that  a  vacuum  may  be  maintained  in  the  latter  by 
means  of  an  air  pump. 

Collodion  sacs  are  sometimes  used  in  animal  experiments.  Bouil- 
lon cultures  are  placed  within  the  sacs,  which  are  then  inserted  in 
the  abdomen  of  an  animal  and  left  there.  The  sac  is  made  of 
collodion  because  it  is  non-absorbent  and  allows  the  bacterial  juices 


NUTRIENT  MEDIA 


lOI 


and  products  to  osmose  outward  and  be  absorbed  by  the  animal, 
while  the  animal  fluids  percolate  into  the  sac.  There  are  several 
very  ingenious  ways  of  making  these  sacs,  but  the  details  are  too 
elaborate  to  be  described  here. 

BOUILLON. 


Bouillon  or  broth  is  the  most  useful  of  all  the  nutrient  media, 
since  it  is  not  only  used  as  a  liquid  medium,  but  by  the  addition 
of  gelatine,  or  agar,  it  is  converted  into  solid 
media. 

There  are  two  methods  of  making 
bouillon. 

Method  I. 

Take  500  grams  of  lean  beef  free  from 
all  fat,  chop  it  fine  and  cover  with  1,000 
c.c.  of  water,  shake  and  place  on  the  ice 
over  night.  Then  squeeze  the  fluid  out  of 
the  meat  by  means  of  a  cloth,  and  supply 
enough  water  to  make  a  litre.  Inoculate 
this  meat  juice  with  a  fluid  culture  of  the 
colon  bacillus  for  the  purpose  of  ferment- 
ing the  meat  sugar.  For  this  purpose  the 
inoculated  juice  is  allowed  to  stand  at 
room  temperature  over  night.  Bring  to  a 
boil  and  add 


10  grams  of  Witte's  peptone. 
5  grams  common  salt. 


Fig.  27. — Kitasato  fil- 
ter   for    filtering    toxins. 
(Williams.) 
Weigh  the  saucepan  and  contents  and  heat 

to  60°  C.     Supply  the  water  lost  by  evaporation.     Neutralize  either 

by  adding  sufficient  sodium  hydrate,  10  percent  solution,  until  red 

litmus  paper  is  colored  a  faint  blue,  or  else  titrate  10  c.c.  of  the 

mixture  with  a  decinormal  solution  of  sodium  hydrate,  using  phenol- 


I02  BACTERIOLOGICAL   LABORATORY   TECHNIC 

phthalein  as  an  indicator,  and  after  finding  how  much  of  a  nor- 
mal solution  is  required  to  neutralize  990  c.c.  (1,000  c.c. — 10  c.c. 
used  for  titration)  this  normal  solution  is  added.  The  mixture 
thus  neutralized  is  then  boiled  for  five  minutes  and  the  weight  re- 
stored. After  boiling,  from  .5  percent  to  1.5  percent  normal  hydro- 
chloric acid  solution  is  added  and  the  acidity  thus  produced  is 
spoken  of  as  -I-.5  per-cent  or  +  i-5  percent  as  the  case  may -be. 

Upon  boiling,  the  albumins  are  coagulated  by  heat,  and  the 
phosphates  are  thrown  down.  The  acid  re-dissolves  the  latter. 
The  former  must  be  removed  by  filtration.  The  filtrate  is  a  clear 
straw-colored  fluid  of  an  acid  reaction  which  should  not  become 
cloudy  upon  boiling.  This  is  then  run  into  flasks  or  test-tubes  and 
sterilized. 

The  second  method  is  much  more  convenient,  and  is  prepared 
by  adding  3  grams  of  Liebig's  beef  extract  to  a  litre  of  water,  and 
adding  the  peptone  and  salt,  as  in  the  previous  method,  and  pro- 
ceeding as  before.  To  filter  the  bouillon,  the  filter  paper  must 
be  folded  many  times,  and  the  funnel  must  be  carefully  cleaned. 

GELATINE. 

To  make  gelatine,  bouillon  is  made  to  which  gelatine  is  added  in 
order  to  render  it  solid.     The  following  steps  are  taken: 

a.  Take  a  litre  of  water  in  a  saucepan  and  add  chopped  beef  or 
beef  extract  as  in  bouillon.  After  standing  over  night  squeeze 
the  beef  and  extract  the  juice. 

b.  Add  I  percent  peptone,  5  percent  salt,  10  percent  to  15  per- 
cent best  gelatine  and  weigh. 

c.  Heat  until  ingredients  are  all  dissolved. 

d.  Neutralize,  gelatine  is  highly  acid  and  requires  much  alkali. 

e.  Boil  five  minutes  and  restore  weight,  boil  till  albumin  coagu- 
lates. 

f.  Cool  to  60°  C.  and  add  an  egg  well  beaten  up  in  water. 

g.  Boil   slowly  till  all  the  egg  is  coagulated.     This  clears  the 


AGAR-AGAR  I03 

medium  of  fine  particles  that  are  not  removed  by  filtration. 
Add  .5  percent  normal  hydrochloric  acid. 

h.  Filter  through  absorbent  cotton  on  a  funnel  previously  wet 
with  boiling  water. 

i.  Tube  and  sterilize  in  autoclave  for  fifteen  minutes  at  110°  C. 
Litmus,  or  lacmoid,  or  neutral  red  may  be  added  to  the  gela- 
tine as  an  indicator. 

AGAR-AGAR. 

To  make  agar : 

a.  Take  20  grams  of  powdered  or  chopped  agar. 

b.  Add  to  500  c.c.  of  water,  place  in  a  can  in  autoclave  and  heat 
to  120°  C.     Then  cool. 

c.  Add  this  to  500  c.c.  of  bouillon  of  double  strength,  making 
1,000  c.c. 

d.  Neutralize. 

e.  Cool  to  60°  C. 

f.  Add  an  egg  to  the  mixture,  stir. 

g.  Boil  till  egg  is  coagulated  thoroughly. 

h.  Titrate  and  adjust  to  desired  acidity  as  given  under  bouillon, 
and   while  boiling  hot,  filter  through  absorbent  cotton  wet 
with  boiling  water, 
i.    Run  into  tubes.     Sterilize.     Slope  the  tubes  for  twelve  hours 

and  store  in  dark  place. 
To  make  glycerine  agar  add  6  percent  of  glycerine  to  the  agar 
before  neutralizing.     To  make  agar  for  tubercle  bacilli,  veal  bouillon 
must  be  employed,  and  glycerine  must  be  added. 

Litmus  Milk. 

Carefully  skimmed  milk,  to  which  litmus  has  been  added,  is  run 
into  tubes  and  sterilized.  This  is  a  valuable  culture  medium.  It 
is  also  a  reagent. 


I04 


BACTERIOLOGICAL  LABORATORY  TECHNIC 


Potato  Tubes. 


I.  Wash  some  large  potatoes  and  with  a  Ravenel  potato  cutter, 
cut  out  semi-cylinders  of  potato.  Immerse  in  running  water  over 
night,  in  order  to  prevent  them  from  turning  black.  It  is  well 
to  wash  these  bits  of  potato  with  i-io,ooo  bichloride 
of  mercury  6  hours  and  running  water  over  night. 
Some  laboratories  soak  their  slices  in  sodium  carbon- 
ate solution.  It  is  desirable  to  know  the  reaction 
of  the  medium  and  each  batch  should  be  tested,  then 
marked  whether  faintly  or  strongly  acid  or  alkalin. 

Thrust  absorbent  cotton  to  the  bottom  of  the  tube 
and  wet  with  distilled  water;  place  the  potato  upon 
the  cotton,  then  plug  the  tube  and  sterilize  in  auto- 
clave twice.     The  tubes  should  be  sealed. 

PEPTONE  SOLUTION— Dunham. 


Take  Peptone lo  grams 

Salt 5  grams 

Water i,ooo  c.c. 

Mix.  Boil.     Filter  and  store  in  tubes  and  sterilize. 

This  is  used   to   demonstrate   the  production  of 

indol. 

_       ^     ^  Dextrose    and    lactose    culture    media   are   often 

Fig.  28. — Po-  1      rT.1  1,11- 

tato  in  culture  used.     They  are  prepared  by  addmg  i  percent  of 

tube.     (Wil-  these  sugars  to  the  various  media  before  neutraliza- 
liams.)  .  "^ 

tion. 


BLOOD  AGAR. 


Is  prepared  by  adding  to  agar  some  defibrinated  rabbit's  blood 
in  varying  proportions  before  the  agar  is  tubed  and  hardened. 


THE    STUDY   OF   THE    GROWTH   OF   BACTERIA  I05 

BLOOD  SERUM. 

The  blood  of  a  dog  drawn  under  strictly  aseptic  precautions 
from  a  vein  of  an  anesthetized  dog  is  collected  in  a  sterile  jar  and 
after  the  serum  has  separated,  it  is  run  into  tubes  by  sterile  pipettes 
and  simply  coagulated  by  heat.  Sterilization  is  not  necessary,  and 
is  harmful  for  the  growth  of  the  tubercle  bacilli,  because  salts  are 
formed  which  interfere  with  the  growth  of  the  bacteria. 

LOFFLER'S  BLOOD  SERUM  MIXTURE. 

Blood  serum  of  an  ox  or  a  horse  is  employed,  mixed  with  bouillon 
containing  i  percent  of  grape  sugar. 

Seventy-five  percent  of  blood  serum  is  mixed  with  25  percent 
bouillon.  This  is  run  into  sterilized  tubes  and  the  latter  are  placed 
in  a  blood  serum  coagulator  and  coagulated  in  a  sloping  position 
at  a  temperature  of  65°  C.  or  thereabouts. 

After  they  are  coagulated  they  are  sterilized  by  heating  an  hour 
each  day  at  65°  C.  five  successive  days,  or  at  95°  C.  for  an  hour  on 
three  successive  days.  After  sterilization  the  tubes  should  be  sealed 
carefully. 

Egg  are  employed  as  culture  media.  The  yolks  and  whites  of  a 
number  of  eggs  are  shaken  together  in  a  flask  and  then  strained 
through  a  towel  to  remove  the  froth.  The  mixture  is  then  run 
into  tubes  and  coagulated  and  sterilized  like  blood  serum.  On 
this  mixture  the  tubercle  bacillus  grows  very  well. 

These  are  the  common  culture  media  used  in  laboratories.  For 
a  more  technical  description  of  the  manufacture  of  these  and  other 
media,  the  student  is  referred  to  books  devoted  to  laboratory  technic. 

Litmus  tincture  is  made  by  adding  a  large  handful  of  litmus  cubes 
to  a  pint  of  water  and  boiling  down  to  one-fourth  its  volume.  This 
is  then  filtered  through  paper  and  stored  after  sterilization. 

The  Study  of  the  Growth  of  Bacteria.— Cultures. 

Bacteria  growing  in  groups  on  culture  media  are  spoken  of  as 
colonies.     Aerobic  bacteria  may  be  made  to  grow  on  culture  media 


io6 


BACTERIOLOGICAL  LABORATORY  TECHNIC 


by  simply  inoculating  the  media  with  some  pus  or  blood  containing 
them,  by  means  of  a  sterile  pipette  or  platinum  needle.  Bouillon 
may  be  thus  inoculated,  as  may  any  of  the  media,  and  other  cultures 
may  be  made  from  these  by  sterilized  needles.  But  such  cultures 
are  made  up  of  colonies  of  different  sorts  of  bacteria — some  patho- 
genic, some  non-pathogenic,  etc.  To  separate  the  various  bacteria 
so  that  they  will  grow  in  isolated  groups,  is  a  comparatively  easy 


Fig.  29, — Colonies  in  gelatine  plate  showing  how  they  may  be  separated  and 
the  organisms  isolated.     (Williams.) 


matter,  and  is  accomplished  in  several  ways.  The  simplest  is  to 
employ  several  tubes  of  agar  or  blood  serum.  Over  the  surface  of 
each  of  these,  a  platinum  loop  containing  pus,  or  other  matter,  is 
rubbed  successively.  These  tubes  are  then  incubated.  After  a 
few  hours,  the  first  one  exhibits  a  copious  growth  of  many  different 
kinds  of  bacteria  growing  confluently  together,  from  which  it  is  im- 
possible to  isolate  any  pure  cultures.  The  second  tube  is  less  covered 
with  bacteria,  while  the  third,  instead  of  containing  a  mass  of  bac- 


THE   STUDY   OF  THE   GROWTH  OF   BACTERIA  I07 

teria,  exhibits  tiny  little  dots,  or  colonies  (pure  cultures)  growing 
discretely  isolated.  By  means  of  a  sterilized  platinum  needle  these 
little  colonies  may  be  fished  out  and  transplanted  to  fresh  culture 
tubes,  and  after  a  few  hours'  growth  they  become  pure  cultures. 


Fig.  30 — Series  of  stab  cultures  in  gelatine,  showing  modes  of  growth  of  different 
species  of  bacteria.     (Abbott.) 

An  old  method  employed  in  many  laboratories,  in  breweries  and 
originated  by  Pasteur  was  what  is  known  as  the  dilution  method. 
Numerous  flasks  are  inoculated  by  matter  containing  bacteria 
very    highly    diluted    in    bouillon    and    by    means    of    a    sterile 


io8 


BACTERIOLOGICAL  LABORATORY  TECHNIC 


O 


pipette  drops  of  this  highly  attenuated  mixture  are  dropped  into 
flasks  of  sterilized  bouillon  or  wort.  Among  a  great  number  of 
flasks  so  inoculated,  some  will  be  found  sterile,  others  will  contain 
two  or  three  different  forms  of  bacteria,  while  a  few  will,  perhaps, 
contain  a  pure  colony  of  the  kind  of  bacteria  for  which  a  search  is 
being  made. 

Another  method  is  to  inject  some  matter  con- 
taining pathogenic  bacteria  into  a  rabbit  or 
guinea  pig.  The  various  juices  and  the  leuco- 
cytes of  the  animal  destroy  the  non-pathogenic 
bacteria  and  a  pure  culture,  often  of  a  pathogenic 
form,  may  be  isolated  from  the  blood  or  miliary 
abscess  or  tubercle  of  the  animal  at  autopsy  and 
transferred  to  culture  media. 

By  far  the  most  useful  and  ingenious  method 
of  procedure  is  the  Koch,  or  plate  method.  This 
is  used  in  many  laboratories  all  over  the  world. 

Koch  was  the  first  to  employ  solid  culture  media 
for  this  purpose,  and  his  method  depends  upon 
the  principle  that  a  liquid  culture  media  may  be 
inoculated  with  bacteria  and  then  spread  out  on 
sterile  glass  plates  or  dishes  where  it  quickly 
hardens,  the  bacteria  being  uniformly  separated 
from  each  other,  and  for  a  time  at  least  kept 
isolated  by  means  of  the  solid  media,  and  after 

Fig.  31. Needles   they  have  developed  into  isolated  colonies  they 

used  for  inoculating  jnay  be  transplanted  to  tubes,  of  media  in  which 
they  may  be  stored.  In  another  way  if  a  man 
wanted  to  secure  a  pure  lot  of  seed  of  a  single  variety  from  a  multi- 
tude of  many  kinds,  it  would  perhaps  be  impossible  to  pick  out  by 
hand  the  seed  wanted  because  of  their  fewness  and  smallness,  but 
if  he  sowed  them  and  waited  until  the  plants  developed  they  could 
then  be  identified  and  gathered  (Abbott).  Thus  it  is  with  plate 
cultures. 


THE  STUDY  OF  THE  GROWTH  OF  BACTERIA      I09 

To  isolate  a  pure  culture  of  bacteria,  say  the  Bacillus  pyocyaneus 
from  pus,  the  following  procedure  is  adopted  in  this  method. 

Three  sterilized  petri  dishes,  and  three  tubes  of  gelatine  melted 
at  40°  C.  are  used.  A  loopful  of  pus  is  taken  up  by  a  sterilized 
platinum  loop  and  mixed  with  the  gelatine  of  the  first  tube.  To  do 
this  the  tube  is  held  across  the  left  hand  in  a  horizontal  position  and 
the  cotton  plug  is  removed,  and  held  by  its  outside  end  between  the 
fingers  of  the  left  hand,  care  being  taken  to  prevent  the  tubal  part 
of  the  plug  touching  anything  and  being  contaminated.  The  plati- 
num loop  is  then  slowly  and  carefully  introduced  into  the  medium, 
and  stirred  around  so  that  the  tube  walls  are  not  touched.  The 
needle  is  again  sterilized  and  tube  number  two  is  held  in  the  palm 


Fig.  32. — Method  of  inoculating  culture  media.     (Williams.) 

of  the  left  hand  parallel  to  the  first  one  and  its  plug  is  removed  also ; 
then  with  a  carefully  sterilized  needle,  three  loops  of  the  inoculated 
gelatine  are  removed  from  number  one  and  mixed  with  number 
two  tube.  The  needle  is  then  again  carefully  sterilized  in  the  flame, 
the  plug  of  number  one  is  carefully  replaced  and  another  tube, 
number  three,  is  held  in  the  palm  of  the  left  hand  and  its  plug  is 
carefully  removed  and  held  as  the  previous  ones  were.  With  the 
sterilized  loop  three  loopfuls  of  the  gelatine  from  number  two  are 
carefully  introduced  into  number  three  and  the  needle  is  then  steril- 
ized and  put  aside.  The  petri  dishes  should  now  be  laid  on  a 
cold  level  slab,  and  the  contents  of  the  tubes  run  into  the  differ- 
ent dishes.     Tube  number  one  is  taken  first;  the  lip  of  the  tube  is 


110       BACTERIOLOGICAL  LABORATORY  TECHNIC 


Fig.  33. — Dilution  method  of  making  cultures,  i,  Is  first  tube  containing 
great  number  of  colonies;  2,  contains  less  number;  3,  relatively  few. 
(WUliams.) 


ROLL  CULTURE  III 

wiped  with  the  cotton  plug  and  then  held  in  the  flame  to  destroy  all 
bacteria  clinging  to  it.  The  lid  of  a  petri  dish  is  carefully  and  par- 
tially lifted  and  the  contents  of  the  tube  rapidly  and  evenly  poured 
over  the  bottom  of  the  plate,  and  the  lid  quickly  replaced. 

This  procedure  is  followed  with  the  other  tubes,  and  then  the 
plates  or  dishes  are  put  in  a  cool  dark  place,  and  the  tubes  are  put 
into  a  solution  of  bichloride  of  mercury,  or  into  boiling  water. 

The  plates  should  be  examined  from  time  to  time.  After  several 
days  a  perfect  cloud  of  round  colonies  are  seen  in  number  one;  a 
large  number  in  No.  2  and  a  much  fewer  number,  say  fifty,  in  No.  3. 
It  is  an  easy  matter  then  to  pick  out  a  colony  that  is  surrounded  by 
a  bluish  green  halo  and  transfer  it  to  a  tube  of  agar  or  bouillon. 
In  the  case  of  pus  it  is  more  than  probable  that  the  colony  is  that  of 
the  pyocyaneus  bacillus,  and  that  it  contains  nothing  but  these 
bacilli.  It  must  be  studied  in  a  dozen  other  ways,  before  it  is  cer- 
tain that  it  is  this  bacillus,  but  the  preceding  method  is  a  necessary 
primary  step  to  secure  this  organism  in  pure  culture  and  may  be 
taken  as  a  pattern  for  all  plate  methods. 

Agar  plates  are  often  used  since  they  have  this  advantage — they 
do  not  melt  at  37°  C.  incubator  temperature.  When  agar  is  used  it 
must  be  melted  at  100°  C.  and  cooled  below  45°  C.  and  above  39°  C. 
Above  45°  C.  bacteria  may  be  killed.  Below  39°  C.  the  agar  begins 
to  harden,  so  this  method  must  be  performed  quickly;  the  plates 
should  be  slightly  warmed,  the  culture  poured  on  and  the  agar  hard- 
ened, they  then  must  be  inverted  in  the  incubator,  since  the  water 
of  condensation  forming  in  the  lids  of  the  plates  often  falls  and 
washes  one  colony  into  another. 

When  gelatine  plates  are  made,  they  must  be  kept  in  a  cool  place. 
It  is  often  of  advantage  to  cool  the  plates  by  means  of  ice,  before 
they  are  filled. 

Roll  Culture. 

Instead  of  pouring  out  the  contents  of  the  inoculated  tubes  the 
gelatine  may  be  made  to  harden  on  the  walls  of  the  tubes  by  quickly 


112 


BACTERIOLOGICAL   LABORATORY  TECHNIC 


rotating  the  tube  in  a  groove  melted  in  a  block  of  ice.  The  centrifu- 
gal force  distributes  the  gelatine  over  the  glass,  and  the  ice  hardens 
it  rapidly  while  in  contact  with  the  glass.  Such  tubes  are  veritable 
plates,  and  in  them  colonies  of  bacteria  often  grow  as  well  as  on  the 
plates  and  may  be  fished  out. 

The  various  characteristics  of  bacterial  growth  may  be  studied  in 
cultures.  Bacteria  differ  in  very  many  ways  in  cultures.  Some 
grow  rapidly  and  luxuriantly;  some  discretely  and  slowly;  colors 
and  odors  are  produced  by  some;  gelatine  is  liquefied  by  many, 
while  others  do  not  liquefy  gelatine.     Milk  is  curdled  and  digested 


Fig.  34. — Esmarchs'  method  of  making  roll  cultures  on  ice.     (Williams.) 


by  some;  gas  and  acids  produced  by  others.     These  various  char- 
acteristics enable  us  to  identify  and  differentiate  bacteria. 

The  cultivation  of  bacteria  in  the  laboratory  has  for  its  purpose 
a  demonstration  of  their  vital  activities.  This  may  indicate  only 
their  botanical  character  or  it  may  show  their  relation  to  disease. 
In  order  that  we  may  classify  germs  systematically  certain  criteria 
have  been  established  which  when  added  together  permit  us  to 
identify  and  name  the  organisms.     This  is  called  determinative 


ROLL   CULTURE  II3 

bacteriology.  The  principal  characters  to  be  noted  are  complete 
morphology,  staining  characters,  particularly  with  Gram's 
method,  colonial  growth  on  agar  and  gelatine,  potato,  blood  serum, 
milk,  sometimes  inorganic  salt  solutions,  the  enzymic  products 
as  indicated  by  fermentation  of  carbohydrates  and  solution  of 
proteins  like  milk  curd  and  gelatine.  With  this  last  comes  ammonia 
and  nitrite  productions.  The  optimum  temperature  and  media 
and  resistance  to  physical  and  chemical  agencies  must  be  taken 
into  consideration.  For  pathogenic  bacteria  we  establish  as  far 
as  possible  the  relations  with  lower  animals.  This  includes,  of 
course,  the  production  of  soluble  toxins  and  endotoxins. 

The  chemical  activities  of  many  bacteria  are  well  displayed  in 
milk  culture.  Milk  is  run  into  tubes,  and  sterilized  tincture  of 
litmus  is  often  added  to  act  as  an  indicator.  Before  using  the  milk, 
it  must  be  skimmed  and  free  from  all  fat. 

The  property  of  converting  sugar  into  acids  and  gases  is  best 
studied  in  fermentation  tubes. 

Into  sterile  fermentation  tubes  bouillon  containing  sugar  is  run, 
these  are  plugged  and  sterilized.  They  may  be  inoculated  with 
bacteria  and  if  gas  production  occurs  it  is  quickly  manifested  in  the 
closed  arm.  The  component  gases  may  be  studied  and  the  various 
properties  determined.  This  gas  ratio  is  of  use  in  identifying 
various  bacteria  and  differentiating  them.  The  closed  arm  of  the 
tube  being  shut  off  from  free  air  by  the  amount  of  bouillon  in  the 
open  arm  is  practically  an  anaerobic  tube  and  is  employed  for  this 
purpose.  Bacteria  that  grow  in  the  closed  arm  are  considered 
anaerobes.  By  inoculating  a  gelatine  tube  with  bacteria  while  it  is 
melted  and  then  letting  it  solidify,  previously  shaking  the  tube 
vigorously,  gas  formation  will  be  speedily  manifested  by  the  presence 
of  bubbles.  Acids  are  detected  in  cultures  by  the  employment  of 
various  indicators  in  the  culture  media.  Litmus,  lacmoid,  and 
neutral  red  are  used  for  this  purpose.  By  titrating  bouillon  of 
previous  known  acidity  with  a  decinormal  soda  solution,  the  amount 
of  acid  produced  by  different  bacteria  can  be  estimated. 
8 


114       BACTERIOLOGICAL  LABORATORY  TECHNIC 

Various  sugars  are  fermented  by  bacteria,  and  lactic,  acetic, 
and  butyric  acids  are  produced.  Indol  is  also  produced  by  many 
bacteria  (colon  bacillus,  cholera  bacillus) ,  and  its  presence  in  culture 
is  an  important  means  of  identifying  different  bacteria.  The 
organism  to  be  studied  must  be  grown  in  culture  media  known  to 
be  free  from  indol.     For  this  purpose,  all  meat  extracts  must  be 


Fig.  35.— Fermentation  tube.     (Williams.) 

excluded  and  a  simple  solution  of  peptone  and  salt,  run  into  tubes 
and  sterilized,  is  used.  After  bacteria  have  grown  in  this  media  for 
several  days  the  indol  produced,  if  it  is  produced,  is  detected  by 
adding  a  few  drops  of  pure  sulphuric  acid.  If  a  red  color  (nitroso- 
indol)  is  not  produced,  a  few  drops  of  sodium  nitrite  solution 
(.02  grams  to  100  c.c.  of  water)  must  be  added,  and  if  a  pink  to  deep 
red  color  does  appear  it  may  be  safely  assumed  that  indol  is  present. 
Ammonia  is  detected  in  culture  by  suspending  a  piece  of  paper 


ROLL   CULTURE  II5 

wet  with  Nessler's  reagent  above  a  bouillon  culture  of  a  given  or- 
ganism.    If  a  yellow  to  brown  color  is  produced  ammonia  is  present. 

Nitrites  are  detected  by  growing  the  organism  in  a  solution  of  a 
nitrate  (see  other  works  for  description). 

Incubate  for  a  week  and  then  add  one  cubic  centimeter  each  of 
the  following  solutions: 

a.  Sulphuric  acid . .5  grams. 

Acetic  acid. 150      c.c. 

b.  Amido  naphthaline i  gram. 

Water 20      c.c. 

Boil,  filter,  and  add  180  c.c.  of  dilute  acetic  acid. 

If  nitrites  are  present  a  pink  color  is  produced  by  these  reagents. 
Enzymes  may  be  detected  by  noting  whether  gelatine  is  liquefied, 
or  milk  curd  digested.  Both  these  actions  are  evidences  of  the 
presence  of  enzymes. 

Bacteria  growing  exclusively  in  the  absence  of  oxygen  are  known 
as  anaerobes;  to  cultivate  these  successively  various  forms  of  appa- 
ratus are  necessary. 

The  following  methods  are  pursued  in  ordinary  laboratory  man- 
ipulations. 

1.  Exclusion  of  oxygen. 

2.  Exhaustion  of  oxygen  by  means  of  an  air  pump. 

3.  Absorption  of  oxygen  by  means  of  chemicals  that  absorb  oxy- 
gen from  the  air.  A  mixture  of  pyrogallic  acid  and  sodium  hydrate 
absorbs  oxygen  rapidly,  leaving  nitrogen  only  in  the  chamber. 

4.  Displacement  of  air  by  means  of  an  air-pump  and  allowing 
hydrogen  to  enter  the  vacuum. 

Under  the  Jirst  method  we  may  either  exclude  oxygen  by  laying 
sheets  of  sterile  mica  or  a  cover-glass  on  the  surface  of  the  agar 
or  gelatine  plates  (Fig.  36),  thus  excluding  air,  or  deep  punctures 
may  be  made  in  tubes  half  filled  with  gelatine  or  agar,  for  growths 
often  occur  in  the  depths  of  the  medium,  especially  if  the  latter  has 
been  boiled  previously  to  expel  the  oxygen;  or,  instead  of  mica, 
sterile  paraffine  may  be  poured  over  the  top  of  the  tube.     The 


Il6       BACTERIOLOGICAL  LABORATORY  TECHNIC 

layer  of  paraffine  excludes  the  air.  Flasks  filled  with  bouillon,  or 
tubes  filled  with  bouillon,  or  melted  agar  may  be  inoculated  with  an 
anaerobic  culture,  but  the  filling  of  the  vessel  with  the  medium  must 
be  absolute  so  that  no  space  is  left  for  air,  otherwise  the  organisms 
may  not  grow.  Roux  employs  a  long  sterile  glass  tube,  which  he 
completely  fills  with  melted  agar  inoculated  with  the  organism  he 
wishes  to  grow.  The  ends  of  the  tube  are  then  sealed  in  a  bunsen 
flame  and  there  being  no  air,  anaerobic  conditions  are  fulfilled. 


Fig.  36. — ^A  streak  made  in  agar  by  a  needle  inoculated  with  anaerobic  bacilli 
and  then  covered  at  one  spot  with  cover-glass.     (Williams.) 


and  organisms  grow.     After  colonies  appear  the  tube  is  broken  at 
a  file-mark  near  the  colony  and  tubes  inoculated  therefrom. 

Under  other  methods  large  Novy  jars  are  used  for  the  reception 
of  petri  dishes  and  test-tubes.  From  these  jars  the  air  is  withdrawn, 
and  hydrogen  allowed  to  flow  into  it.  A  solution  of  pyrogallic  and 
sodium  hydrate  is  placed  in  the  bottom  of  the  jar  to  absorb  any 


ANIMAL  EXPERIMENTS 


117 


remaining  oxygen.  There  are  many  other  ingenious  mechanical 
ways  of  growing  bacteria  under  anaerobic  conditions  and  the  student 
is  referred  to  works  devoted  entirely  to  technic. 


Fig.  37. — Novy  jar. 


Animal  Experiments. 


To  determine  the  pathogenicity  of  bacteria;  to  measure  the 
strength  of  toxins  and  anti-toxins,  to  standardize  anti-toxins,  and  to 
recover  bacteria  in  pure  culture,  it  is  often  imperative  that  small 
laboratory  animals  be  used.  Guinea  pigs,  rabbits,  and  mice  are 
oftenest  employed.  Strong  young  animals  are  the  best.  Culture 
toxins  and  pathological  material  are  intoduced  into  their  bodies 
in  various  ways.  A  favorite  one  is  to  shave  the  abdomen,  scour  it 
with  soap  and  water,  and  then  bichloride  of  mercury,  and  finally 
sterile  water.  With  a  pair  of  sterile  scissors  a  small  hole  is  cut  in 
the  abdominal  parieties  and  through  it  a  loop  containing  a  drop  of 


Il8       BACTERIOLOGICAL  LABORATORY  TECHNIC 

culture  is  run  into  the  peritoneal  cavity,  or  under  the  skin.  The 
animal  is  carefully  weighed,  and  it  is  watched  from  day  to  day. 
If  it  dies  an  autopsy  is  made  on  it. 

Other  methods  consist  in  injecting  fluid  culture  into  the  veins  of 
the  ear,  or  into  the  peritoneum,  by  means  of  a  sterile  hypodermic 
syringe.  The  autopsy  should  be  made  carefully ;  the  animal  should 
be  thoroughly  wet  with  a  solution  of  bichloride  of  mercury,  then  it 
should  be  stretched  over  a  pan,  especially  devised  for  the  purpose, 
or  nailed  to  a  board.  The  skin  over  the  abdomen  and  thorax  must 
then  be  shaved  and  sterilized  with  a  solution  of  bichloride  of  mer- 
cury. The  walls  should  then  be  seared  in  a  line  from  the  throat  to 
the  pubes  with  a  hot  knife,  and  through  this  line  a  cut  should  be 
made  opening  up  the  thoracic  and  abdominal  cavities. 

By  means  of  a  hot  knife  spots  must  be  seared  on  the  various  organs, 
and  with  another  sterile  knife  cuts  should  be  made  into  the  organs, 
then  through  these  cuts  sterile  platinum  needles  are  thrust,  and 
then  culture  media  are  inoculated  with  them.  Sometimes  it  is  neces- 
sary to  remove  bits  of  tissue  from  various  organs  and  place  them 
in  culture  media.  In  the  recovery  of  the  tubercle  bacillus  from 
animals  this  procedure  is  necessary.  Great  care  must  be  taken  in 
making  the  culture  and  all  tubes  should  be  carefully  stored.  Often 
it  is  of  great  importance  to  make  smears  on  cover-slips  as  well 
as  cultures,  from  the  heart  cavities,  liver,  kidney,  peritoneal  cavity, 
etc.,  and  stain  them  directly  with  Jenner's  stain.  It  is  sometimes 
necessary  to  inject  cultures,  or  bits  of  nerve  tissue  from  a  rabies  case 
into  the  brain.  To  do  this,  remove,  under  strict  aseptic  precautions, 
a  button  of  bone  from  the  skull  by  means  of  a  trephine. 

Histological  Methods. 

Sections  of  tissues  from  infected  animals  are  often  examined  and 
stained  by  appropriate  methods.  To  demonstrate  bacteria,  the 
tissues  should  be  hardened  in  absolute  alcohol,  and  imbedded  in 
celloidin,  then  cut  into  sections  and  mounted  in  the  following 
different  ways: 


HISTOLOGICAL   METHODS  II 9 

I.  Loffler's  Method. 

a.  Float  section  in  alcohol. 

b.  Remove  with  section  lifter  to  Loffler's  methylene  blue  from  five  to  thirty 

minutes. 

c.  Decolorize  in  i  percent  solution  of  acetic  acid  for  ten  seconds. 

d.  Dehydrate  in  absolute  alcohol  for  a  few  minutes. 

e.  Clear  in  xylol. 

f.  Mount  in  balsam. 

II.  Weigert's  Method. 

a.  Transfer  section  to  alcohol. 

b.  Place  in  lithium  carmine  five  minutes. 

c.  Then  in  acid  alcohol  fifteen  seconds. 

d.  Wash  in  water. 

e.  Transfer  to  slide  and  dry  with  blotting  paper. 

f.  Apply  Ehrlich's  gentian  violet  for  three  minutes. 

g.  Blot  and  place  in  Gram's  solution  for  two  minutes, 
h.  Wash  and  dehydrate  in  aniline  oil. 

i.    Wash  with  xylol. 

j.    Dry,  mount  in  balsam  and  examine. 

In  Loffler's  method  all  the  tissues,  especially  the  nuclei  and  the 
bacteria,  appear  blue. 

In  Weigert's  method,  if  the  bacteria  stain  by  Gram's  method, 
the  tissues  appear  pink,  the  bacteria  a  deep  blue-black.  This  lat- 
ter method  is  an  admirable  one.  There  are  many  other  methods 
of  staining.  Paraffine  embedding  methods  may  be  employed,  but 
for  these  the  student  is  referred  to  works  solely  devoted  to  technic. 
The  staining  methods  are  the  same  for  paraffine  and  in  experienced 
hands  give  better  results. 


CHAPTER  VII. 

ANTISEPTICS  AND  DISINFECTANTS. 

Many  chemical  substances  have  the  power  of  entering  into  chem- 
ical union  with  the  protoplasm  of  bacterial  cells  and  so  forming  new 
compounds,  and  often  coagulating  the  protoplasm. 

Bacteria  differ  in  their  powers  to  resist  these  agencies;  the  anthrax 
spore  is  much  more  difficult  to  kill  than  the  typhoid  bacillus;  these 
chemical  substances  act  at  a  high  rather  than  a  low  temperature. 

A  chemical  disinfectant,  such  as  copper  sulphate,  acts  more 
rapidly  and  effectively  in  a  watery  solution  than  in  a  complex 
albuminous  one. 

It  is  often  necessary  to  determine  the  exact  minimum  amount  of 
an  antiseptic  that  will  destroy  a  given  organism  or  produce  a  com- 
plete inhibition  of  growth;  for  this  purpose  small  amounts  of  a  dis- 
infectant are  added  to  gelatine  in  test-tubes  and  these  are  poured 
into  plates  and  the  result  noted. 

Previous  to  pouring  the  plates  each  tube  is  inoculated  with  a 
loopful  of  culture  and  thoroughly  mixed  with  the  medium. 

Another  method  is  to  make  bouillon  cultures  of  an  organism  and 
add  to  each  a  certain  percentage  of  the  solution  of  the  antiseptic, 
and  abstract  every  few  minutes  after  the  addition  of  the  chemical 
one  loopful  of  the  mixture  and  inoculate  fresh  media. 

It  will  be  found  in  the  case  of  most  antiseptics  in  dilute  solution 
that  an  interval  of  time  must  elapse  before  the  organisms  are  killed. 
This  is  determined  by  observing  the  cultures  made  from  the  mixture. 
After  five  minutes,  growth  may  occur,  but  after  one  hour,  all  may  be 
dead,  or  it  may  take  two  or  three  hours. 

The  most  valuable  chemical  disinfectants  are  those  that  kill  in 
highly  dilute  solution  in  a  short  time. 

I20 


CHEMICAL   DISINFECTANTS  121 

Pieces  of  thread  sterilized,  and  then  put  in  fluid  cultures  may 
be  used  in  experiments;  they  are  dipped  into  solutions  of  chemicals 
for  varying  lengths  of  time  and  then  placed  in  culture  media  and 
growth  noted. 

Bichloride  of  mercury  is  a  highly  efficient  germicide  in  watery 
solutions;  if,  however,  albuminous  matter  is  present  its  action  is 
inhibited  very  much. 

CHEMICAL  DISINFECTANTS. 

Mercury  Salts. — Bichloride  of  mercury  in  highly  dilute  solution 
is  a  very  valuable  antiseptic.  It  dissolves  in  i6  parts  of  hot  water. 
It  requires  an  acid  reaction  for  most  favorable  action  and  the 
tablets  now  on  the  market  are  made  up  with  some  acid  having  no 
effect  upon  the  mercury  salt.  In  i-ioo  watery  solution  this  salt 
will  kill  anthrax  spores  in  twenty  minutes.  In  blood,  the  anthrax 
bacillus  is  killed  by  a  1-2,000  solution  in  a  few  minutes.  In  bouillon 
the  same  organism  is  killed  in  a  dilution  of  1-40,000;  in  water, 
1-500,000;  all  in  the  same  interval  of  time.  The  presence  of  the 
albumins  in  the  blood  or  bouillon,  no  doubt  acts  as  a  protecting 
envelope  about  the  bodies  of  the  bacteria.  Bichloride  is  then  more 
efficient  outside  the  body  than  in  it.  It  is  also  more  useful  and 
powerful  when  it  is  acidulated  with  a  .  5  percent  of  HCl,  or  when  it 
is  mixed  with  common  salt  or  ammonium  chloride.  In  culture 
1-1,000,000  solution  prevents  the  growth  of  most  pathogenic  bac- 
teria. Biniodide  of  mercury  is  said  by  some  observers  to  be  more 
powerful  than  the  bichloride.  It  is  certainly  less  likely  to  be  inter- 
fered with  by  albumins. 

Sulphate  of  copper  in  water  is  a  powerful  germicide.  It  is  more 
potent  in  watery  solution  than  in  bouillon.  It  has  a  remarkable 
affinity  for  algae  and  for  moulds.  The  author  found  that  if  moulds 
are  put  into  alkaline  solution  of  copper  sulphate  and  heated,  the 
copper  enters  into  chemical  union  with  the  protoplasm  of  the 
mycelia,  hyphae,  and  the  spores;  1-400,000  of  copper  sulphate  in 


122  ANTISEPTICS  AND   DISINFECTANTS 

water  destroys  the  typhoid  bacilli.  Even  nascent  copper  kills  the 
typhoid  bacilli,  so  that  copper  foil  in  drinking  water  has  the  power, 
after  a  few  hours  contact,  of  destroying  bacteria  in  the  water. 

The  silver  salts  are  useful  in  medicine  as  disinfectants,  especially 
on  mucous  surfaces.  The  nitrate  of  silver  is  one  of  the  most  valu- 
able of  all  the  preparations;  it  is  about  a  fourth  as  efficient  as  bi- 
chloride of  mercury  and  is  not  nearly  so  toxic.  Some  of  the  albu- 
minates of  silver  are  useful  because  of  their  non-irritating  action. 

Acids,  especially  the  mineral  ones,  are  valuable  disinfectants  in 
not  too  dilute  solutions.  They  act  chiefly  as  inhibitors  of  growth 
rather  than  destroyers  of  bacterial  cells.  In  the  healthy  stomach, 
hydrochloric  acid  acts  as  a  normal  disinfectant,  and  in  disease, 
where  it  is  absent,  it  must  be  added  in  order  to  prevent  decomposi- 
tion of  food.  Boric  acid  is  useful  in  medicine  on  mucous 
membranes. 

The  halogens,  iodine,  bromine  and  chlorine,  are  active  agents 
for  the  destruction  of  bacteria.  The  cheapest  of  these  is  chlorine. 
It  acts  best  in  contact  with  moisture,  since  it  decomposes  the  mole- 
cule of  water  combining  with  the  hydrogen  to  form  free  HCl  and 
setting  free  oxygen. 

Dry  chlorine  gas  (45  percent)  failed  to  kill  dry  anthrax  spores  in 
one  hour,  but  when  moisture  was  introduced  4  percent  chlorine 
killed  the  spores. 

Chloride  of  lime,  chlorinated  lime,  in  i  percent  solution  kills  most 
bacteria  in  1-5  minutes.  Iodine  preparations  like  chlorine  ones 
are  very  powerful.  They  are  of  great  use  in  medicine;  ordinary 
tincture  of  iodine  painted  over  infected  areas  acts  as  a  powerful 
germicidal  agent.  It  is  too  expensive  to  use  in  house  disinfection 
and  it  is  exceedingly  destructive  to  all  metallic  objects.  A  5  percent 
solution  in  50  percent  alcohol  acts  as  a  splendid  disinfectant  for 
intrauterine  injection  in  puerperal  sepsis.  It  is  now  said  that  10 
percent  iodine  tincture  in  70  percent  alcohol  is  the  most  efficacious, 
practical,  medical  disinfectant.  Many  claim  it  to  have  the  highest 
penetrating  powers. 


CHEMICAL  DISINFECTANTS  1 23 

Carbolic  acid  is  valuable  as  a  disinfectant  because  of  its  stability. 
A  1-1,000  solution  inhibits  bacterial  growth;  a  ,5  percent  solution 
kills  spores  in  a  few  hours.  A  thorough  solution  should  be  made, 
and  to  be  very  efficient,  5  percent  HCl  should  be  added  to  it. 

Cresol,  lysol  and  creolin  are  useful  as  disinfectants,  but  are 
sometimes  unreliable  since  perfect  solution  cannot  always  be  made. 
The  mixture  of  one  of  these  substances  with  water  is  more  of  an 
emulsion  than  solution.  Anthrax  spores  have  been  known  to  live 
for  hours  in  creolin  solutions.  The  value  of  these  cresols  is  that 
when  applied  to  a  surface  the  water  may  evaporate  but  the  germi- 
cide sticks  and  continues  its  effects.  Glycerin  is  sometimes  added 
to  lighter  phenol  solutions  to  assist  this  action. 

Peroxide  of  hydrogen  has  a  great  reputation  in  medicine  as  an 
antiseptic.  It  kills  bacteria,  especially  the  pus  cocci,  in  a  few 
minutes  in  a  15  percent  solution.  A  40  percent  solution  will  kill 
anthrax  spores  in  a  few  hours.  It  is  a  powerful  agent  when  fresh, 
and  is  not  poisonous.  It  combines  with  organic  matter  and  becomes 
inert.  It  degenerates  if  exposed  to  atmosphere  and  if  it  comes  in 
contact  with  the  ferments  of  the  blood  (haemase). 

Formaldehyde  gas,  CHjO,  is,  by  all  means,  the  most  useful,  as 
well  as  the  most  powerful  disinfecting  agent  that  we  have.  In 
solution  40  percent  in  water,  it  is  known  as  formaline.  It  has  a 
marked  affinity  for  organic  substances  and  forms  chemical  combi- 
nations with  many  organic  bodies.  When  it  unites  with  ammonia 
it  becomes  inert  until  some  acid  frees  it.  It  unites  with  iron,  but 
other  metals  are  unaffected.  Its  use  in  medicine  is  wide  and  varied. 
It  is  a  deodorizer;  renders  gelatine  glass-like  and  insoluble  in  boiling 
water.  It  may  be  liberated  as  a  gas  in  apartments  and  ships, 
actively  destroying  all  bacteria.  One  percent  of  the  vapor  in  the 
air  of  a  closed  room,  if  the  air  is  moist,  destroys  bacteria  after 
twelve  hours.  It  is  best  to  keep  the  room  closed  for  twenty-four 
hours.  It  may  be  thrown  into  the  room  in  many  ways ;  by  genera- 
tors which  decompose  the  vapor  of  wood  alcohol,  when  they 
reach  hot  platinum  sponges,  salt,  or  hot  copper;  by  vaporizing  a 


124  ANTISEPTICS  AND   DISINFECTANTS 

solution  by  means  of  heat;  by  adding  permanganate  of  potash  to  a 
solution  of  formaline;  by  spraying  a  concentrated  solution  over 
bedding,  floors,  and  walls,  then  closing  the  apartment.  It  is  very 
much  more  active  in  warm  air  than  in  cold,  and  when  the  air  is 
moist.  It  has  been  known  to  destroy  anthrax  spores  wrapped  up 
in  paper  and  placed  under  blankets.  All  of  the  pathogenic  bacteria 
are  killed  by  it,  the  Staphylococcus  aureus  and  anthrax  spores  being 
more  resistant  than  anything  else.  It  will  not  kill  moulds  unless 
highly  concentrated.  As  dilute  watery  and  alcoholic  solutions 
decompose  they  should  only  be  used  when  freshly  made. 

Sulphur  Dioxide  Gas. — An  old  and  rather  unreliable  form 
of  disinfectant.  It  does  not  kill  anthrax  spores  very  readily,  as 
it  requires  an  exposure  of  twenty-four  hours  to  a  40  percent  vapor 
in  a  room.  It  is  generated  by  burning  sulphur  in  a  room  tightly 
closed,  and  it  is  much  more  efficient  if  water  is  vaporized  in  the 
room.  It  is  not  very  penetrating,  is  poisonous  to  breathe,  speedily 
bleaches  fabrics,  and  attacks  metal  objects.  It  is  much  superior  to 
formaline  as  an  agent  for  the  destruction  of  insects,  especially 
mosquitoes,  also  to  kill  rats  infected  with  plague  bacilli. 

Lime. — Ordinary  thick  lime,  or  whitewash,  is  highly  germicidal. 
It  is  especially  efficacious  in  disinfecting  feces  from  typhoid  cases. 
Typhoid  bacilli  are  killed  after  one  hour's  exposure  to  a  20  percent 
mixture. 

Potassium  permanganate  in  3  percent  solution  is  said  by  Koch 
to  kill  anthrax  spores  in  twenty-four  hours.  It  is  not  so  efficient  a 
germicidal  agent  as  supposed. 

Turpentine  and  essential  oils  are  efficient  germicides  in  con- 
centration. Common  mustard  rubbed  in  the  hands  is  said  to  make 
them  sterile. 

Alcohol. — Ninety-five  percent  and  absolute  alcohols  are  not 
antiseptic  for  the  anthrax  spores,  since  they  will  live  for  many  hours 
in  contact  with  absolute  alcohol.  In  general  it  is  unreliable. 
Seventy  percent  alcohol  is  the  most  efficient  strength. 

Zinc  chloride  in  concentration  is  a  powerful  germicide.     A  2 


ANTISEPTIC  VALUES  1 25 

percent  solution  will  kill  the  ordinary  pyogenic  bacteria  in  two 
hours. 

Sputum,  urine  and  dejecta  are  best  disinfected  by  heat.  Chem- 
icals often  are  inert  because  they  cannot  penetrate  the  albuminous 
masses  of  the  sputum  or  feces.  Long  contact  with  carbolic  acid 
acidulated  with  HCl  is  very  efficient.  Concentrated  formaline  and 
solutions  of  chloride  of  lime  may  be  used,  also  a  heavy  mush  of 
lime  in  water. 

Boiling  or  heating  instruments  and  dressings  by  high  moist  heat, 
as  in  an  autoclave,  is  the  most  reliable  method  of  rendering  them 
sterile.  The  exposure  of  dressings  to  150°  C.  for  one  hour,  or  boil- 
ing instruments  for  twenty  to  thirty  minutes  makes  them  certainly 
sterile. 

Disinfection  of  the  skin  is  a  difficult  undertaking  from  a  bacterio- 
logical standpoint.  In  the  deep  layers  of  the  skin,  and  in  the  sweat 
glands  and  hair  follicles,  bacteria  often  exist,  even  after  the  most 
thorough  and  prolonged  disinfection.  The  application  of  soap  and 
water  with  a  stiff  brush  is  by  all  means  the  most  valuable  part  of  the 
process,  since  with  the  removal  of  the  dirt  most  of  -the  bacteria  are 
removed.  Thorough  scrubbing  with  soap  and  sterile  water, 
followed  by  scrubbing  with  a  1-1,000  bichloride  solution,  cleansing 
the  nails  with  a  sterile  brush,  and  prolonged  immersion  in  bichloride 
or  permanganate  of  potash  solution,  complete  the  process.  Modern 
methods,  even  after  all  this  preparation,  require  the  use  of  rubber 
gloves  that  have  been  sterilized  by  boiling.  The  faultiest  part  of 
the  preparation  for  an  aseptic  operation  from  a  bacteriological 
standpoint,  has  always  been  considered  to  be  the  sterilization  of 
the  hands,  and  if  these  can  be  covered  by  rubber  gloves  that  are 
sterile,  the  fault  can  be  surely  eliminated. 

Antiseptic  Values.     (After  Park.) 

The  figures  refer  to  the  relative  antiseptic  powers  of  various  agents 
for  fluids  containing  organic  matter. 

Alum I  to      222 

Aluminium  acetate i  to    6,000 


126  ANTISEPTICS  AND   DISINFECTANTS  • 

Ammonium  chloride i  to  9 

Boric  acid i  to       143 

Calcium  chloride i  to         25 

Calcium  hypochlorite i  to    1,000 

Carbolic  acid i  to       333 

Chloral  hydrate i  to       107 

Copper  sulphate i  to    2,000 

Ferrous  sulphate i  to       200 

Formaldehyde,  40  percent i  to  10,000 

Formaldehyde,  pure i  to  20,000 

Hydrogen  peroxide,  fresh i  to  14,300 

Mercuric  chloride i  to  40,000 

Mercuric  iodide i  to  25,000 

Quinine  sulphate i  to       800 

Silver  nitrate i  to  12,500 

Zinc  chloride i  to       500 

Zinc  sulphate i  to         20 


CHAPTER  V. 

BACTERIA. 

STREPTOCOCCUS  PYOGENES. 

Streptococcus  Pyogenes. 

Streptococcus  Erysipelatis.     Chain  Coccus.     (Fig.  38.) 
Morphology  and  Stains. — Cocci  grow  in  catenate  form  of  from 
4  to  40  individuals  to  a  chain.     Each  coccus  comprises  two  hemi- 


FiG.  38. — Streptococcus  pyogenes.     (Kolle  and  Wassermann.) 

spheres  divided  transversely.  Some  chains  appear  branched.  The 
cocci  are  not  motile,  and  do  not  have  spores.  They  can  be  stained 
with  all  basic  stains,  and  are  not  decolorized  by  Gram's  method. 

Relation  to  Oxygen. — They  grow  either  in  the  presence  or 
absence  of  oxygen,  and  are,  therefore,  facultative  aerobes. 

Temperature  and  Food  Requirements. 

Develop  best  at  37°  C.     Will  not  grow  at  47°  C.     Never  vegetate 

127 


128  BACTERIA 

luxuriantly  on  any  culture  media,  but  are  most  prolific  on  one  that 
is  faintly  acid  and  contains  animal  juices  like  serum.  They  must  be 
transplanted  frequently.  On  gelatine  they  grow  scantily  without 
liquefaction,  the  growth  consists  of  discrete  little  masses,  while 
on  agar  with  glycerine,  they  appear  translucent  colonies  of  very 
small  grayish  granula.  In  bouillon  cultures  some  varieties  either 
cloud  the  medium  uniformly,  or  else  sedimentate  in  the  form  of 
little  balls,  the  supernatant  fluid  remaining  clear.  It  ferments  some 
simple  sugars  but  does  not  form  gas.  In  milk  the  growth  is  more 
luxuriant,  and  becoming  acid,  is  totally  coagulated  in  twenty-four 
hours.  Clotted  casein  may  be  digested.  On  potato  the  growth 
is  invisible  and  scanty. 

Vital  Resistance. — Thermal  death-point  is  54°  C.  in  five 
minutes.  Virulence  in  dried  albuminous  matter  (pus)  is  retained 
for  months.  If  kept  on  ice,  vitality  and  virulence  are  retained 
for  months  also. 

Chemical  Activities. — Lactic  acid  and  sulphuretted  hydrogen  are 
produced,  also  ferments  which  have  the  property  of  dissolving  fibrin 
under  anaerobic  conditions.  They  are  also  capable  of  dissolving 
red  blood  corpuscles,  either  in  culture  media  or  in  the  body  and 
about  cultures  on  blood  agar  plates  there  is  a  clear  halo  of  hemolysis. 
They  produce  a  strong  soluble  toxin,  which  can  be  filtered  from  the 
bouillon  and  precipitated  with  alcohol.  This  causes  necrosis, 
anaemia  and  death. 

Habitat. — In  sewage,  dwellings,  dust,  on  the  healthy  human  body, 
and  in  the  cavities  of  the  respiratory  tract,  vagina,  rectum,  and  in 
the  feces.  It  is  the  cause  of  many  diseases,  i.e.,  erysipelas,  puerperal 
fever,  meningitis,  pneumonia,  endocarditis,  peritonitis,  tonsillitis, 
osteomyelitis,  and  the  diarrhoea  of  children. 

In  general  septicaemia  streptococcus  is  found  in  the  blood,  and 
plays  an  important  role  in  secondary  infection,  causing  an  aggrava- 
tion of  the  original  infection,  and  often  death.  It  is  especially  active 
in  phthisis,  scarlatina,  small-pox,  and  diphtheria,  in  which  diseases 
it  is  often  the  cause  of  death.     Many  of  the  symptoms  of  phthisis 


PNEUMOCOCCUS  1 29 

are  due  to  the  toxins  of  the  streptococcus;  cavity  formation  and  hectic 
fever  for  example.  Its  virulence  can  be  intensified  by  passing  it 
through  a  series  of  animals,  until,  finally,  yxmr  ^^  ^  cubic  milli- 
meter killed  in  one  day  all  the  mice  injected  with  this  dose.  The 
toxin  contains  a  peculiar  haemolytic  substance,  which,  as  before 
remarked,  dissolves  red  cells  of  the  blood,  hence  the  anaemia  in  sep- 
ticaemia and  in  suppuration.  The  toxin  of  the  streptococcus,  if  in- 
jected under  the  skin,  causes  redness  like  erysipelas.  Coley's  fluid 
containing  this  toxin  is  used  to  treat  sarcomata,  since  infection  with 
the  streptococcus  has  been  known  to  cause  a  disappearance  of  these 
tumors.     Practically  all  animals  are  susceptible  to  the  streptococcus. 

Agglutinations. — The  serum  from  an  animal  injected  with  strep- 
tococci, or  immunized  against  it,  will  agglutinate  streptococci. 

Anti-toxic  sera  have  been  prepared  by  injecting  horses  .with  highly 
virulent  living  culture  of  streptococci.  The  serum  protects  to  a 
limited  degree,  and  has  some  curative  properties.  Cultures  of  cocci 
from  human  sources  have  been  found  to  produce  the  best  toxins; 
there  are,  however,  many  strains.  ") 

PNEUMOCOCCUS. 

Streptococcus  lanceolatus,  commonly  known  as  the  pneu- 
mococcus,  or  Diplococcus  lanceolatus.     (Fig.  39.) 

Morphology  and  Stains. — This  organism  is  usually  found  in  the 
tissues  and  sputum,  in  the  form  of  lance-shaped  cocci,  surrounded 
by  a  capsule.  Is  almost  always  associated  in  pairs,  though  some- 
times in  chains  of  five  or  six  members.  In  albuminous  fluids,  or 
blood  serum,  and  in  milk,  the  organism  exhibits  a  well  defined  cap- 
sule; in  bouillon  and  other  media,  it  loses  the  capsule  and  the  lan- 
ceolate shape,  and  often  appears  spherical,  in  pairs,  or  chains.  It 
is  not  motile,  has  no  flagella  or  spores,  is  easily  stained  by  all  the 
basic  aniline  dyes,  and  keeps  its  color  by  Gram's  method.  Under 
certain  conditions  it  strongly  resembles  the  streptococcus  pyogenes, 
and  may  be  differentiated  therefrom  by  growing  it  on  agar  smeared 
9 


I30 


BACTERIA 


with  blood.  The  streptococcus  causes  a  haemolysis  of  the  corpuscles, 
while  the  pneumococcus  does  not  and  the  colonies  are  greenish. 

Oxygen  Relations. — It  is  a  facultative  aerobe. 

Grows  rapidly,  but  never  luxuriantly  at  37.5°  C;  at  22°  C.  much 
more  slowly,  often  not  at  all.  Grows  better  in  the  presence  of 
serum  or  hemoglobin. 


Fig.  39. — Diplococcus  pneumoniae,  from  the  heart's  blood  of  a  rabbit.     X 1000 
(Frankel  and  Pfeiffer.) 

Vital  Resistance. — Easily  killed  at  a  temperature  of  52°  C,  ex- 
posed for  ten  minutes.  Direct  sunlight  also  kills  it  in  twelve  hours. 
While  it  quickly  dies  on  ordinary  culture  media,  it  may  live  in  dried 
sputum  or  pus  exposed  to  diffuse  sunlight  and  desiccation,  for  four 
months. 

Cultures. — On  gelatine  plate  it  produces  very  minute  colonies 


PNEUMOCOCCUS  I3I 

after  quite  a  length  of  time.  On  glycerine  agar  it  grows  better, 
but  the  colonies  are  small  and  difficult  to  see.  In  both,  the  colonies 
are  whitish,  with  a  pearly  lustre.  On  blood  serum  it  grows  in  trans- 
parent colonies.  In  bouillon  it  grows  feebly,  with  a  whitish  sedi- 
ment, and  in  the  form  of  chains.  Here  the  growth  is  inhibited  by 
the  products  of  its  own  metabolism,  i.e.,  lactic  acid.  If  this  is  neu- 
tralized by  putting  chalk  into  the  bouillon  the  growth  becomes 
luxuriant  and  the  bouillon  becomes  thick.  On  potato  it  will  not 
grow.  It  ferments  some  of  the  sugars,  the  most  important  being 
inulin.     No  gas  is  formed. 

Habitat. — Outside  the  human  body  it  has  not  been  found,  but  is 
normally  present  in  the  mouth  of  about  30  percent  of  all  people. 
Even  the  alveoli  of  the  lungs  in  health  contain  them.  Human  saliva 
injected  into  animals  often  causes  pneumococcic  siepticaemia.  They 
are  also  found  on  the  conjunctiva  and  nose  in  health. 

Chemical  Activities. — No  soluble  toxin  has  been  discovered. 
The  toxic  properties  are  due  to  an  endo-toxin.  This  organism  is 
a  pyogenic  one,  and  causes  dense  fibrinous  exudates  on  serous 
membranes.  All  tissues  of  the  body  may  be  attacked.  Some 
strains  of  pneumococci  are  more  neurotoxic  than  others. 

In  rabbits  an  injection  (intravenous)  of  pneumococci  very  often 
(33  percent)  causes  lobar  pneumonia;  certain  strains  cause  lobular 
pneumonia  habitually  among  the  susceptible  animals  (Eyre).  In 
human  infection  the  organisms  are  forcibly  inhaled  into  the  deepest 
recesses  of  the  lungs.  Pneumonia  may  be  haematogenous  in  origin 
also. 

Besides  pneumonia,  any  serous  membrane  may  be  attacked  and 
pleuritis,  peritonitis,  pericarditis,  or  meningitis  may  be  caused. 
Abscesses  anywhere  may  be  due  to  the  pneumococcus.  Mucous 
membranes  of  the  throat  often  "are  affected;  middle  ear  abscesses 
also  may  be  caused  by  this  organism.  Pneumococcic  septicaemias 
are  common. 

During  pneumonia,  pneumococci  may  be  recovered  from  the 
blood  before  the  crisis  by  means  of  blood  cultures;  10  c.c.  of  blood 


132  BACTERIA 

abstracted  from  veins  is  mixed  with  500  c.c.  of  milk  and  incubated. 
In  twenty-four  hours  pneumococci,  if  present,  grow  luxuriantly. 
Just  before  the  crisis  the  organisms  will  not  grow. 

Immunity  and  Susceptibility. — The  susceptibility  of  man  varies 
greatly.  Exposure  to  cold  and  hardships  of  various  kinds  predis- 
pose to  pneumonia.  One  attack  does  not  prevent  another.  It  has 
been  observed  that  normal  leucocytes  only  become  phagocytic 
toward  the  pneumococcus  when  lying  in  anti-pneumococcic  serum. 
It  has  even  been  noticed  that  these  organisms  grow  better  in 
the  anti-serum,  rather  than  in  the  normal  serum.  Animals  have 
been  immunized  by  injecting  cultures  and  toxin.  The  immune 
serum  thus  produced  protects  small  animals  against  infection,  and 
stimulates  phagocytosis.  It  has  been  used  therapeutically  in  man 
for  the  cure  of  pneumonia  with  doubtful  results.  Oleate  of  soda 
aids  in  bacteriolysis  of  pneumococci  by  sera,  if  added  to  the 
various  varieties  of  immune  sera. 

Agglutination  of  pneumococci  is  caused  by  the  blood  of  infected 
individuals,  even  diluted  at  1-60.  Immune  serum  also  has  the 
same  action. 

Opsonins  increase  during  the  course  of  pneumonia  and  are  at 
their  height  at  or  just  after  crisis. 

Two  intermediary  streptococci  are  Str.  viridans  and  Str.  mucosus, 
Str.  viridans  is  like  the  Str.  pyogenes  but  produces  germ  colonies. 
It  is  most  frequently  met  as  the  cause  of  valvular  endocarditis. 
Str.  mucosus  is  a  long  chain  former  surrounded  by  a  halo  not  stain- 
able  as  a  capsule  and  produces  viscid  exudate.  In  some  ways  it 
resembles  the  pneumococcus. 

The  various  streptococci  from  pus,  saliva,  feces,  manure  and  sew- 
age are  differentiated  by  their  action  on  blood,  milk  and  the  sugars. 

COCCUS  OF  MENINGITIS. 

Streptococcus  Intracellularis. 

Diplococcus  intracellularis  meningitidis. 
Meningococcus.     (Fig.  40.) 


coccus   OF   MENINGITIS  1 33 

This  organism  is  the  cause  of  cerebro-spinal  meningitis. 

Morphology  and  Stains. — Resembles  the  gonococcus  closely, 
because  it  grows  in  biscuit  shaped  pairs;  is  nearly  always  within  pus 
cells,  and  like  the  gonococcus  it  is  decolorized  by  Gram's  stain. 


Fig.  40. — Meningococcus  in  spinal  fluid.     (From  Hiss  and  Zinsser's  Bacteri- 
ology, Copyright  by  D.  Appleton  &  Co.) 

In  reality  it  is  a  micrococcus,  because  it  divides  in  two  planes. 
It  has  no  spores  or  flagella;  is  not  motile;  grows  in  short  chains  at 
times,  and  on  ordinary  media  best  at  37°  C.     It  is  Gram  negative. 

Relation  to  Oxygen. — It  is  an  obligate  aerobe. 

Vital  Resistance. — It  is  killed  after  10  minutes'  exposure  to  65° 
C.  and  is  easily  destroyed  by  drying,  and  by  light.  It  dies  out 
rapidly  on  artificial  culture  media. 


134  BACTERIA 

Cultures. — On  glycerine  agar  it  grows,  sparingly  as  white  viscid 
colonies;  occasionally  it  develops  on  potato;  thrives  on  blood 
senun,  especially  if  smeared  with  blood,  and  does  not  liquefy  the 
serum. 

Habitat. — It  is  found  in  the  pus  from  the  meninges,  sputum, 
and  nasal  mucus  of  persons  afflicted  with  epidemic  meningitis, 
or  spotted  fever.  It  has  been  found  in  the  mucous  membranes  of 
healthy  individuals,  and  these  persons  may  be  ** carriers"  of  infec- 
tion. After  spinal  puncture,  it  may  be  seen  in  the  pus  cells,  and 
the  diagnosis  of  the  disease  can  be  made  in  this  way. 

Virulence. — It  is  scarcely  virulent  for  lower  animals.  If  given 
by  hypodermics  into  the  pleura,  or  peritoneum,  it  produces  death  in 
mice.  Meningitis  may  be,  in  monkeys,  produced  by  sub-dural 
injection. 

Chemical  Activities. — ^Produces  an  endo-toxin  buf  no  soluble 
toxin.     It  is  not  chromogenic. 

Agglutination  is  caused  by  immune  serum. 

Method  of  Infection. — The  infection  atrium  of  the  coccus  is 
not  certainly  known  but  most  of  the  evidence  points  to  the  nasal 
passages  and  cribriform  plate  to  the  sub-dural  space. 

Specific  Therapy. — Flexner  and  Jobling  have  produced  an  anti- 
serum for  meningitis.  It  has  anti-bacterial  powers.  Horses  are 
injected  with  bacterial  suspensions  until  their  serum  possesses 
curative  properties.  This  anti-serum  is  injected  directly  into  the 
arachnoid  space  by  lumbar  puncture,  after  withdrawal  of  some 
of  the  meningitic  exudate.  Little  anti-serum  will  appear  in  the 
cerebro-spinal  fluid  if  it  be  injected  subcutaneously.  Therapeutic 
results  have  been  brilliant. 

There  is  another  important  Gram  negative  diplococcus  in  the 
nose  called  Micrococcus  catarrhalis.  It  is  differentiated  from 
the  meningitis  organism  by  its  free  yellow  growth  on  agar  and 
absence  of  active  pathogenic  properties.  It  is  thought  to  have 
some  relation  to  acute  coryza. 


STAPHYLOCOCCUS  PYOGENES  AUREUS 

STAPHYLOCOCCUS  PYOGENES  AUREUS. 


135 


Staphylococcus  Pyogenes  Aureus.     (Fig.  41.) 

Micrococcus  Pyogenes. 

Staphylococcus  ^pyogenes  aureus,  albus,  and  citreus  are  known 
commonly  as  staphylococcus,  or  grape  coccus.  They  differ  only 
in  color  production  on  artificial  media. 


Fig.  41. — Staphylococcus  aureus.     (Williams.) 

The  Micrococcus  pyogenes  aureus  only  is  here  described. 

Morphology  and  Stains. — Round  cocci,  often  growing  in 
bunches  like  grapes.  Individual  cocci  dividing  in  two  planes. 
They  stain  very  well  with  all  basic  dyes,  and  are  not  decolorized  by 
Gram's  method.  They  are  not  motile;  have  neither  flagella  nor 
spores. 

Oxygen  Requirements. — The  coccus  grows  well  in  oxygen, 
and  poorly  without  it. 

Temperature  and  Vital  Resistance.^ — Thrives  best  at  body 
temperature,  but  grows  well  at  room  temperature.     Resists  drying 


136 


BACTERIA 


for  over  one  hundred  days  in  pus.  Dry  thermal  death-point  is  80° 
C.  for  one  hour.  Moist  heat  70°  C,  kills  in  ten  to  twenty  minutes. 
Resists  freezing  temperature  for  many  months. 

Exceedingly  resistant  to  formaldehyde,  more  so  than  some 
spore-bearing  organisms.  Resists  light  also. 
It  is  killed  by  corrosive  sublimate  i-iooo 
in  1 5  minutes;  i  percent  HjOj  in  30  minutes. 
Chemical  Activities. — Produces  a  golden 
yellow  pigment  only  under  oxygen.  Gen- 
erates acids,  but  no  free  gases.  Creates 
indol  and  sulphuretted  hydrogen;  ferments 
urea,  and  produces  ferments  that  dissolve 
gelatine,  and  the  coagulated  proteids  of 
milk.  The  toxin  is  soluble  in  water,  and 
acts  intensely,  causing  violent  local  reaction. 
If  in  the  abdominal  cavity,  it  causes  perito- 
nitis. Subcutaneously  it  may  produce  sterile 
abscess,  or  local  necrosis.  There  is  pro- 
duced in  cultures  a  toxin  having  a  destruc- 
tive action  upon  leucocytes  and  red  blood 
cells. 

Cultures.T^In  gelatine  it  rapidly  forms 
golden  yellow  colonies,  that  quickly  liquefy 
the  gelatine.  (Fig.  42.)  Sterile  products 
of  the  growth  also  liquefy  gelatine.  On 
gelatine  plate,  yellowish  to  orange  colonies 
Fig.  42.— Gelatine  cul-  are  formed.     On  agar  streak  a  luxuriant 

rS'Sd.lwuila"™)  "^-"g-^  g-^"-*  develops.  In  bouillon  there 
is  a  marked  even  cloudiness,  with  a  fine 
pellicle  on  surface;  moderate  sediment,  which  upon  shaking  is 
broken  up.  Milk  is  rendered  acid  and  curdles  very  soon,  the  curd 
being  digested  finally. 

Potato  cultures  are  dry,  whitish,  then  yellow,  and  finally  deep 
orange. 


GONOCOCCUS  137 

Habitat. — Widely  distributed;  found  in  dirty  water,  sewage,  air, 
dust  of  streets  and  houses;  also  upon  the  skin;  normally  present 
in  the  mouth,  nose,  rectum,  anterior  urethra,  vagina,  and  external 
ears. 

Pathogenesis. — In  man  it  is  the  cause  of  carbuncles,  abscesses, 
osteomyelitis,  septicaemia,  puerperal  infection,  and  any  inflamma- 
tion of  the  serous  membranes.  It  causes  acne  and  boils;  can,  and 
does  attack  any  tissue  of  the  body.  Endocarditis  is  a  very  grave 
affection  that  is  caused  by  this  organism.  It  also  plays  an  important 
role  in  secondary  infection,  causing  necrosis  of  previously  infected 
tissues  (tubercles)  and  is  active  in  small-pox  and  diphtheria.  Ex- 
perimental endocarditis  has  been  produced  in  animals  by  injecting 
it  into  the  veins.  By  passage  through  animals  it  is  rendered  highly 
virulent.  In  young,  diabetic  and  anaemic  subjects,  its  action  is 
often  rapidly  fatal.  Its  pathogenic  action  is  often  wide  and  disas- 
trous. By  growing  it  under  anaerobic  conditions  its  Virulence  may 
be  intensified,  and  the  activity  with  which  it  liquefies;  gelatine  is  an 
index  of  its  malignancy. 

In  man  acne,  boils,  and  carbuncles  have  followed  the  rubbing  of 
culture  into  the  skin. 

Immunity. — Thus  far  it  has  been  impossible  to  produce  any 
marked  immunity  either  by  anti-toxic  sera,  or  by  culture,  living  or 
dead,  but  the  bacterins  made  from  this  germ  have  been  used  with 
excellent  results  in  all  but  the  very  aggravated  and  fulminating 
affections  caused  by  it.  Bacterin  treatment  of  acne  and  furunculo- 
sis  has  established  itself  as  most  efficacious. 

There  is  a  member  of  this  group  infesting  the  deep  layers  of  the 
skin  called  Micro,  epidermidis  albus.  It  is  of  feeble  pathogenic 
power,  but  may  delay  the  healing  of  surgical  wounds. 

GONOCOCCUS. 

.  Micrococcus  Gonorrhoeae  (Neisser). 
Diplococcus  Gonorrhcece,  commonly  called  the  gonococcus.    (Fig.  43.) 


138  BACTERIA 

Morphology  and  Stains. — The  morphology  of  this  organism  is 
peculiar  and  characteristic.  Always  found  in  pairs  which  are  ce- 
mented by  an  invisible  substance.  These  pairs  resemble  coffee 
beans  with  the  concave  sides  opposite  each  other  and  slightly  apart; 
or  kidneys  placed  with  the  hilums  facing  each  other. 

In  pus  it  is  generally  found  within  the  pro- 
toplasm of  the  leucocytes,  about,  though  never 
within,  the  nuclei.     It  is  non-motile;  has  no 
flagella,  or  spores,  and  stains  readily  with  all 
the  basic  stains,  but  best  with  Loffler's  blue. 
It  is  decolorized  by  Gram's  stain.     This  point 
is  most  important  in  differentiating  it  from 
Fig.  43. — Gonococci  other  diplococci,  except  the  meningococcus.    A 
ica^^iagnosL^^  ^     ^     diplococcus  is  said  to  exist  normally  in  some 
urethras   that  resembles  the  gonococcus,  but 
is  Gram  positive. 

Oxygen  Requirements. — It  is  a  facultative  anaerobe. 
Vital  Conditions. — It  is  cultivated  with  difficulty  in  culture 
media.  Grows  best  at  about  36°  C.  As  it  dies  quickly  in  usual 
culture  media,  a  special  one  must  be  employed;  that  containing  as- 
citic or  hydrocele  fluid,  blood  or  urine  is  best.  It  does  not  withstand 
high  temperature,  drying,  or  light,  very  long,  and  is  very  easily  killed 
in  culture  by  silver  salts.  In  tissues  of  the  urethra  it  may  live  many 
months. 

Cultures. — On  agar,  containing  ascites  fluid,  it  grows  very  spar- 
ingly. The  colonies  are  exceedingly  delicate,  and  gray,  turning 
to  yellowish,  and  are  scarcely  above  the  culture  media.  It  will  not 
grow  in  gelatine,  milk,  or  ordinary  bouillon,  but  in  one  made  of 
nutrose,  serum,  beef-extract,  and  peptone. 

Habitat. — Never  found  outside  the  human  organism,  except  on 

linen,  towels,  instruments,  etc.     It  is  in  all  senses  a  strict  parasite. 

Bacterial  Activities. — Apparently  does  not  produce  a  soluble 

toxin,  but  an  endo-toxin  (gonotoxin) ,  which  is  highly  resistant  to 

heat. 


MICROCOCCUS  TETRAGENUS  1 39 

Pathogenic  Virulence. — This  organism  does  not  infect  any  of 
the  lower  animals.  The  "  gonotoxin,"  if  injected  into  small  animals, 
produces  a  doughy  infiltrated  area,  which  undergoes  necrosis.  It 
has  been  found  that  filtrates  of  old  cultures  (sterile),  if  placed  on 
urethral  mucous  membranes,  can  produce  suppuration.  In  man, 
the  organism  causes  a  distressing  disease  (gonorrhoea),  which  may 
become  a  dangerous  one,  ending  even  in  death.  It  may  produce  vio- 
lent inflammation  of  the  urethra,  vagina,  uterus,  fallopian  tubes, 
and  the  peritoneum.  It  frequently  affects  the  conjunctivae  of  the 
newly  born,  and  sometimes  causes  a  pan-ophthalmia,  which  destroys 
the  sight.  It  is  a  common  cause  of  suppurating  arthritis  gonor- 
rhoeal  rheumatism,  endocarditis,  pleuritis.  In  fact,  any  serous 
membranes  may  be  infected,  and  very  serious  results  follow.  Cysti- 
tis caused  by  the  gonococcus  is  sometimes  followed  by  infection  of 
the  kidneys.  In  the  urethra,  the  cocci  may  burrow  deep  beneath 
the  epithelial  cells,  and  set  up  a  metaplasia,  or  abscess  formation. 
The  purulent  exudate  is  rich  in  phagocytes  gorged  with  cocci,  often 
as  many  as  40  being  found  within  a  cell. 

Immunity. — One  infection  does  not  confer  immunity  against 
further  infection.  There  is  no  reliable  means  of  producing  artifi- 
cial immunity.  However  gonococcus  bacterins  are  of  some  value 
for  chronic  gonorrhoea.  Torrey  has  been  able  to  obtain  from  rabbits 
an  anti-serum  of  therapeutic  value  in  gonorrhoeal  arthritis. 

MICROCOCCUS  TETRAGENUS. 

Micrococcus  Tetragenus. 

Morphology  and  Stains. — Round  or  oval  cocci;  found  in  pairs; 
more  commonly  in  fours  differing  in  size.  In  culture  this  form  of 
growth  is  apt  to  vary,  and  not  to  be  characteristic.  In  sections  of 
human  or  animal  tissues,  tetrads  only  are  found  that  are  always 
surrounded  by  a  capsule  which  is  stained  easily  by  eosin.  The 
cocci  are  stained  by  Gram's  method.  It  is  not  motile,  and  does  not 
form  spores. 


I40  BACTERIA 

Oxygen  Requirements. — It  grows  very  well  in  the  presence  of 
oxygen,  and  poorly  without  it. 

Cultures. — Grows  well  on  all  common  cuKure  media.  On  gela- 
tine plates  its  growth  is  characterized  by  small  white  colonies, 
elevated,  with  sharp  outlines.  It  does  not  liquefy  the  gelatine.  On 
agar  it  grows  even  more  luxuriantly  than  on  gelatine.  In  bouillon 
it  thrives  well,  depositing  a  heavy  precipitate.  In  milk  it  causes 
coagulation  after  four  days.  On  potato  it  also  grows,  leaving  a 
silvery  streak  where  the  inoculating  needle  was  drawn. 

Chemical  Activities. — It  produces  acid  in  sugar  bouillon,  but 
does  not  form  gas,  indol,  or  H2S. 

Habitat. — Has  never  been  found  outside  the  human  body;  is 
normally  present  in  the  saliva,  sputum  of  tuberculous  subjects,  in 
the  cavities  of  phthisical  lungs,  and  in  abscesses. 

Pathogenesis. — While  causing  a  fatal  septicaemia  in  mice,  and 
abscesses  in  rabbits,  it  is  not  of  much  moment  from  a  pathological 
standpoint,  though  it  plays  an  important  role  in  secondary  infection 
in  phthisis. 

BACILLUS  OF  MALTA  FEVER. 

Bacterium  Melitensis. 

Micrococcus  Melitensis. 

Bacillus  of  Malta  Fever. 

Coccus  of  Malta  Fever. 

An  organism  belonging  somewhere  between  the  Coccacae  and 
Bacteriacae.  It  is  small,  oval-shaped,  and  of  about  .5/1  diameter, 
occurring  in  culture  singly,  in  pairs,  or  in  chains.  In  the  latter 
form,  the  organism  elongates  and  resembles,  more  strongly,  bacilli. 
It  is  non-motile  and  it  has  no  spores.  Stains  faintly  with  the  com- 
mon basic  dyes,  but  not  by  Gram's  method.  It  has  been  found  in 
the  blood  during  life,  and  by  splenic  puncture. 

Cultures. — On  gelatine  its  growth  is  slow,  without  liquefaction. 
On  agar  the  growth,  at  37°  C.,  is  more  rapid.     The  colonies  are 


INFLUENZA  BACILLUS  I4I 

pearly  white,  becoming  yellow.  In  bouillon  it  produces  turbidity, 
with  a  flocculent  deposit.  No  pellicle  is  formed.  On  potato  an 
invisible  growth  occurs.  Milk  is  not  coagulated,  nor  are  acids  or 
gases  produced. 

Pathogenesis. — It  causes  in  man,  Malta  fever.  Rabbits,  guinea 
pigs,  and  mice  are  not  susceptible  to  inoculation,  but  the  disease 
can  be  produced  in  monkeys. 

Agglutination. — The  serum  from  an  individual  suffering  from 
Malta  Fever  agglutinates  the  bacilli,  even  in  dilutions  as  high  as 
I- 1 00. 

Diagnosis  of  the  disease  can  be  effected  by  the  agglutination 
test,  and  by  splenic  puncture,  and  blood  cultures. 

It  is  present  in  the  blood  and  is  excreted  via  the  urine  and  milk. 
The  goat  while  not  suffering  with  malta  fever  can  carry  the  germs 
in  its  body  and  excrete  them  in  the  milk.  Goats'  milk  is  a  general 
food  in  Malta.  The  inference  is  obvious.  Flies  may  transmit  the 
bacilli. 

INFLUENZA  BACILLUS. 

Bacterium  Influenzae. 

Influenza  bacillus . 

Morphology  and  Stains. — ^Very  small  short  rods -which  are  often 
in  pairs,  found  within  epithelial  and  pus  cells,  and  in  sputum;  from 
40  to  80  in  a  cell.  Sometimes  found  chain-like.  No  flagella  or 
spores  are  formed.  Stains  weakly.  Carbol  fuchsin,  diluted,  gives 
the  best  result.  The  ends  of  the  bacillus  stain  more  deeply  than  do 
the  rest  of  the  cell.     It  is  decolorized  by  Gram's  stain. 

Oxygen  Requirements. — It  is  a  strict  aerobe. 

Cultures  grow  best  on  blood  smeared  agar,  or  in  blood  bouillon 
between  27°  C.  and  41°  C. ;  best  at  37°  C.  Blood  or  haemoglobin  is 
required  for  all  cultures.  In  bouillon  it  grows  in  thin  white 
flocculi.  On  agar  in  small  transparent  "dewdrop"  colonies, 
never  luxuriantly.     Grown  in  the  same  culture  with  Staphylococcus 


142 


BACTERIA 


aureus,  it  increases  more  luxuriantly  (symbiosis).  It  is  probable 
that  the  cocci,  in  some  way,  alter  the  blood  of  the  culture  media. 

Vitality. — It  is  easily  killed  by  light,  heat  and  drying.  Lives 
but  a  day  in  distilled  water,  and  from  eight  to  twenty-four  hours  in 
dried  sputum. 

Habitat. — Never  outside  the  body;  always  a  strict  parasite.  It 
is  found  in  the  mucous  membranes  of  the  upper  respiratory  tract, 
and  in  the  mucous  secretions. 

Pathogenesis. — If  pure  cultures  are  placed  on  the  mucous  sur- 
faces of  monkeys,  influenza  results.  Pure  cultures  injected  into  the 
peritoneum  of  guinea  pigs  cause  fatal  peritonitis.  In  man,  it  causes 
various  affections  of  the  upper  respiratory  tract — bronchitis,  pneu- 


FiG.  44. — ^Pest  Bacilli  from  spleen  of  rat.     (KoUe  and  Wassermann.) 

monia,  both  croupous  and  catarrhal.  Also  conjunctivitis.  It  elab- 
orates a  powerful  toxin,  which  produces  strongly  depressing  effects 
on  certain  organs,  especially  nervous  tissues.  It  is  an  important 
factor  in  abscess  production  in  the  middle  ear,  and  elsewhere,  and 
complicates  many  pneumonia  cases,  seriously  interfering  with 
recovery  in  young  children,  and  the  aged.  Associated  with  the 
pneumococcus,  its  toxic  effect  is  increased.  It  has  been  found  in  the 
blood. 


KOCH-WEEKS   BACILLUS  143 

Influenzal  meningitis  is  more  frequent  than  formerly  or  at  least 
is  more  often  diagnosed.  It  can  be  reproduced  in  monkeys. 
Bacilli  appear  in  the  blood  in  influenzal  meningitis. 

By  immunizing  a  goat  with  influenza  bacilli  Wallstein  obtains  a 
serum  which  has  a  pronouncedly  favorable  effect  upon  the  ex- 
perimental disease  in  monkeys  and  promises  some  therapeutic 
power  for  human  beings.  Its  most  important  effect  is  to  stimulate 
phagocytosis  in  the  cerebro- spiral  fluid. 

No  immunity  results  from  infection.  No  leucocytosis  occurs 
during  infection.  Serum  from  infected  individuals  agglutinates 
bacilli  even  if  diluted  1-500. 

No  artificial  immunity  can  be  produced  but  bacterins  are  some- 
times used  therapeutically. 

Bordet-Gengou  Bacillus  of  Whooping  Cough. — This  is  a 
very  minute  ovoid  rod  lying  separately,  varying  from  .8-1. 5/i  long 
and  being  .3//  wide.  No  spores,  no  motility  or  flagella.  Stains 
poorly,  best  at  ends;  Gram  negative.  It  may  be  cultivated  from 
expectoration  early  in  the  disease  upon  media  containing  glycer- 
ine, potato,  blood  and  agar.  Aerobe,  and  grows  best  at  37°  C. 
There  is  an  an  endo-toxin.  Infective  for  monkeys.  The  discoverers 
claim  this  to  be  the  cause  of  pertussis,  because  it  will  act  as  an 
antigen  and  fix  complement  away  from  the  hemolytic  series. 

Conjunctivitis. — There  are  two  specific  germs  for  conjunctivitis 
separate  from  the  gonococcus.  They  are  the  bacillus  of  Koch- 
Weeks  and  that  of  Morax  and  Axenfeld. 

Koch-Weeks  Bacillus. — The  organism  of  pink  eye.  This  is 
a  minute,  i.5/£X.2/i  non-motile.  Gram  negative,  sporeless,  poorly 
staining  rod,  very  like  the  influenza  bacillus.  It  is  aerobic  and  non- 
liquefying.  It  grows  as  minute,  pearly,  glistening,  discrete  colonies, 
only  upon  agar  of  .5  percent  strength. 

The  Bacillus  of  Morax  and  Axenfeld. — A  non-motile,  sporeless 
diplo-  rod;  negative  to  Gram  stain.  Grows  only  in  the  presence  of 
serum  or  blood  and  liquefies  the  former.  It  is  larger  than  the 
Kock- Weeks  bacillus,  measuring  up  to  2^. 


144  BACTERIA 

PLAGUE  BACILLUS. 

Bacterium  Pestis. 

Plague  Bacillus.     (Fig.  44.) 

Morphology  and  Stains. — Short  plump  rods  with  rounded  ends, 
containing  no  spores  and  non-motile.  Also  surrounded  by  cap- 
sule? Organisms  from  exudates,  or  blood,  exhibit  character- 
istically peculiar  polar  staining.  They  are  often  found  within 
the  leucocytes.  In  bouillon  the  organism  grows  in  long  chains;  is 
stained  with  all  the  common  basic  dyes,  but  is  not  colored  by  Gram's 
method  in  cultures.  It  exhibits  a  great  variety  of  involution  forms 
when  grown  in  salty  culture  media  (3 J  percent  salt). 

Relation  to  Oxygen. — Strict  aerobe,  the  growth  is  stopped  by 
the  exclusion  of  oxygen. 

Vital  Requirements. — Grows  well  at  22°  C,  but  best  at  37°  C.; 


Fig.    45. — Colonies    of    plague    bacilli    forty-eight    hours    old.     (Kolle    and 

Wassermann.) 

is  killed  after  a  short  exposure  to  55°  C.-6o°  C.,  stands  drying  from 
four  to  eight  days,  and  dies  in  water  after  a  week.  In  the  buried 
bodies  of  man  and  animals  it  lives  from  twenty-two  to  thirty-eight 


PLAGUE   BACILLUS  145 

days.  Withstands  freezing  for  months,  but  does  not  stand  light 
or  chemicals  very  long. 

Cultures. — Grows  very  well  on  culture  media.  In  bouillon 
it  thrives  abundantly,  with  a  heavy  pellicle  which  produces  depend- 
ent stalactites  that  drop  to  the  bottom  of  the  vessel.  On  gelatine 
plates  it  grows  in  small  flat  colonies,  which  are  gray  and  transpar- 
ent, and  which  do  not  liquefy  the  gelatine.  (Fig.  45.)  In  gelatine 
tubes  it  forms  a  faint  thread-like  line,  without  liquefying  the  media. 
On  agar  the  growth  is  whitish  and  abundant,  and  resembles  the 
colon  bacillus.  Old  cultures  are  luxuriant.  Milk  is  not  coagulated, 
and  the  growth  is  slight.  Potato  yields  a  slow  whitish-yellow 
growth  that  is  sharply  outlined. 

Chemical  Activities. — Does  not  produce  HgS,  enzyme,  colors, 
or  odors,  indol  or  nitrites.     The  toxin  produced  is  not  soluble  and 


Fig.  46. — B.  Pestis  in  pus  of  bubo.     (Jackson.) 

the  filtrate  is  non-poisonous.     Old  killed  bouillon  cultures  can  be 
extracted  and  a  highly  poisonous  substance  precipitated  therefrom 
with  alcohol,  or  ammonium  sulphate,  that  is  lethal  for  mice. 

Habitat. — Never  found  in  healthy  human  bodies.     In  persons 
afflicted  with  plague,  the  organism  is  widely  distributed  in  buboes 
10 


146  BACTERIA 

and  in  the  cutaneous  pustules,  lymphatics  and  in  the  lungs  in  plague 
pneumonia;  more  rarely  in  the  blood  and  other  organs.  In  ani- 
mals, plague  occurs  in  rats.  It  is  supposed  that  some  tropical  soil 
bacilli  infect  rats,  and  becoming  accustomed  to  the  rodent's  body, 
are  eventually  transmitted  to  man.  The  bacilli  are  transmitted 
from  rat  to  rat  in  India  by  the  rat  fleas  which  also  can  bite  man. 
The  organisms  remain  in  the  flea  for  some  time.  Rats  are  also 
infected  from  dead  rats.  In  epidemic  times  the  soil  becomes 
infected  and  persons  going  barefoot  may  be  infected. 

Pathogenesis. — Highly  pathogenic  for  man.     Is   the  cause  of 
the  bubonic  or  Oriental  plague;  bacilli  gain  entrance  by  way  of  the 


Fig.  47. — Pest  bacillus  involution  forms  produced  by  growing  on  3  per  cent 
salt  agar.     (KoUe  and  Wassermann.) 

skin,  causing  localized  foci  of  infection  from  which  buboes  develop, 
followed  by  pest-sepsis  and  death.  The  lungs  may  be  the  original 
site  of  invasion,  and  plague  pneumonia  (worst  form  of  the  disease) 
may  result.  The  typical  bacilli  can  be  found  in  the  sputum  of  the 
patient  thus  affected.  The  mortality  from  this  plague  is  from  50 
percent  to  80  percent.     (Fig.  46.) 

Almost  all  domestic  animals — rats,  mice,  guinea  pigs,  rabbits  and 
squirrels  are  affected;  horses  and  swine  are  very  susceptible;  cows 


147 

and  dogs  less  so.  Rats  seem  to  be  affected  with  a  chronic  form  of  the 
malady,  and  by  inhabiting  ships  and  warehouses  in  foreign  countries, 
spread  the  disease.  Post  mortems  on  infected  animals  reveal  haemor- 
rhagic  petechia  and  serous  infiltration  into  serous  cavities.  Death 
is  generally  due  to  a  profound  toxaemia  and  exhaustion. 

The  virulence  of  the  organism  can  be  raised  by  passing  it  through 
a  series  of  animals. 

Serum  from  infected  animals  agglutinates  plague  bacilli. 

The  diagnosis  of  the  plague  bacilli  is  made  by  rubbing  the  sus- 
pected culture  upon  the  freshly  shaven  skin  of  a  guinea  pig;  if  the 
animal  developes  buboes  and  dies,  and  polar  staining  bacilli  are 
found,  it  is  probable  that  the  organism  is  the  plague  bacillus.  Fur- 
ther, if  curious  involution  forms  develop  on  heavily  salted  agar 
(3  percent)  the  diagnosis  is  confirmed.     (Fig.  47.) 

The  disease  is  spread  by  flies  which,  according  to  Yersin,  are 
susceptible  to  the  disease,  and  spread  it  by  depositing  their  feces 
on  the  human  skin,  rather  than  through  their  bites. 

Immunity. — It  is  possible  to  immunize  against  the  disease. 
Kitasato  and  Yersin  produced  an  anti-toxic  serum,  which  has,  not 
only  a  prophylactic,  but  a  curative  action.  By  the  use  of  killed 
culture  Haffkine  vaccinated  many  people  against  the  plague  very 
successfully. 

FRIEDLANDER'S  BACILLUS. 

Bacterium  Pneumoniae. 

Friedlander' s  Pneumonia  Bacillus.     (Fig.  48.) 

Morphology  and  Stains. — Short  plump  rods  with  rounded  ends, 
surrounded  by  a  thick  gelatinous  capsule  in  animal  fluids,  and  when 
grown  in  milk;  is  not  motile,  and  has  no  spores;  does  not  stain  by 
Gram's  method,  but  easily  by  the  common  basic  dyes. 

Oxygen  Requirements. — Grows  in  and  without  oxygen,  upon 
all  culture  media. 

Chemical  Activities. — ^Produces  abundant  acids,  CO2  and  H. 
gas,  alcohol,  indol,  ferment  and  HjS. 


148  BACTERIA 

Habitat. — Has  been  found  in  soil;  sometimes  in  healthy  saliva. 
Culture  Media. — Grows  luxuriantly  on  all  culture  media. 
On  gelatine  it  grows  in  roundish  elevated  colonies  that  are  yel- 
lowish-white with  a  slimy  lustre,  and  never  liquefies  the  gelatine. 
In  agar  it  multiplies  even  more  abundantly  with  a  moister  growth. 
The  border  of  streak  cultures  is  smooth  and  wavy,  and  the  water 
of  condensation  is  cloudy.  In  bouillon  the  growth  is  very  cloudy 
with  a  silvery  deposit  at  the  bottom.  The 
bouillon  becomes  thickened.  Milk  is  not 
coagulated,  and  potato  yields  a  luxuriant 
yellowish,  moist  shining  growth. 

Pathogenesis. — It  is  possible  to  cause  pneu- 
monia in  mice,  also  septicaemia.  Ouinea 
pigs   and   dogs   are    susceptible.     It  may  be 

.  '^  k"  48--Friedland-  ^^^^^  -^^  normal  mouths.     Friedlander's  pneu- 
er  s  bacillus  and  pneu-  ^ 

mococci,  showing  cap-  monia  is  much  less  frequent  than  that  due  to 

kirDiagnosL"f '  ^^^'  *«  pneumococcus,  but  it  is  very  fatal.  Ag- 
glutination  takes  place  with  immiine  serum. 

This  is  the  most  important  representative  of  a  group  of  organisms 
of  moderate  pathogenic  powers  and  importance  called  variously. 
Bacterium  aerogenes,  Bacterium  mucosus  or  Aerogenes  mucosus 
group.  They  all  have  a  luxuriant  growth  on  media;  are  negative 
to  Gram  stain;  ferment  most  of  the  carbohydrates ;  are  non-motile 
and  most  of  them  show  a  capsule  when  in  the  animal  body. 

Perkins  divides  them  as  follows: 

I.  Bacterium  aerogenes  type  ferments  all  carbohydrates  with  gas. 

II.  Bacterium  pneumoniae  group  ferment  all  carbohydrates  but 
lactose,  with  gas. 

III.  Bacterium  lactis  aerogenes  group  ferment  all  carbodydrates 
except  saccharose,  with  gas. 

These  organisms  are  important  members  of  the  intestinal  flora. 

The  Bacterium  lactis  aerogenes  group  is  a  very  large  one  and 
includes  nearly  all  the  forms  engaged  in  milk  souring.  The 
ordinary  B.  lactici  is  very  like  the  colon  bacillis,  but  is  non-motile. 
It  forms  lactic   acid  among  its  principal  products.     The   most 


TYPHOID    BACILLUS  149 

important  lactic  acid  producer  is  Bad.  bulgaricum  of  Massol. 
This  is  the  principal  ferment  of  the  eastern  sour  milks,  Kumyss 
and  Yoghurt.  Because  of  the  large  amount  of  lactic  acid  formed 
by  this  germ,  Metchnikoff  has  advocated  cultures  of  it  and  sour 
milk  made  by  it  in  the  treatment  of  intestinal  putrefaction  and 
fermentation.  The  Bacterium  bulgaricum  produces  a  soft  milk 
curd  and  an  excess  of  lactic  acid  and  alcohol.  The  bacteria  are 
non- motile,  non-spore  forming.  Gram  positive  and  vary  from  2/1 
to  50/z  in  length.  They  grov^r  with  difficulty  in  the  laboratory,  best 
on  milk  and  whey.  Optimum  temperature  44°  C.  They  form 
branching  filamentous  colonies.  Milk  is  coagulated  in  18  hours 
at  44°  C.  and  in  36  hours  at  37°  C.  The  clot  is  not  dissolved. 
Gelatine  is  not  liquefied. 

TYPHOID  BACILLUS. 

Bacterium  Typhi.     Koch  and  Eberth. 

Bacillus  Typhosus. 

Typhoid  Bacillus.     (Fig.  49.) 

A  most  important  pathogenic  organism  that 
causes  typhoid  fever. 

Morphology  and  Stains. — Generally  short 
plump  rods   i  to  3 // long,  and  .6  to  .8/x  broad. 
Forms  long  threads  in  cultures,  especially  on 
potatoes.     Polar    metachromatic    bodies    are 
sometimes  seen  as  are  unstained  areas  when 
alkaline  methylene  blue  is  used.     The  rod  is      Fig.     49.— Typhoid 
flageHated  (peritrichous) ;  contains  no  spores ;  g^^^^/^  (j^oSrand  Was- 
exhibits  pleomorphic  and  involution  forms;  is  sermann.) 
actively  motile,  and  stains  with  all  the  basic 
aniline  dyes,  but  not  by  Gram's  method. 

Vital  Resistance. — The  thermal  death-point  is  60°  C.,  ten  to 
fifteen  minutes.  Remains  alive  in  ice  for  months;  even  the  tempera- 
ture of  liquid  air  does  not  destroy  it.  In  distilled  water  it  lives  for 
months,  but  if  other  saprophytic  bacteria  are  associated  with  it, 


ISO 


BACTERIA 


however,  it  quickly  dies.  Does  not  resist  drying  or  chemicals, 
except  carbolic  acid,  towards  which  it  exhibits  a  tolerance.  Sun- 
light kills  it  in  an  hour. 

Habitat. — It  never  exists  in  nature,  except  where  water  or  soil 
has  been  contaminated  by  feces  or  urine.  It  may  multiply  in  po- 
table waters,  in  milk,  and  the  juices  of  oysters. 

Chemical  Activities. — Does  not  produce  proteolytic  enzymes; 
forms  HgS.  but  will  not  ferment  the  sugars  with  gas  formation. 
Does  not  yield  indol  or  nitrites.     Produces  levorotatory  lactic  acid. 


Fig.  50. — Seventy-two  hour  old  culture  of  typhoid  bacillus  on  gelatine.     (Kolle 
and  Wassermann.) 

Its  toxin  is  all  contained  within  the  bacterial  cell  (endo-toxins) 
and  is  not  water  soluble.  This  toxin  is  manifested  by  injecting 
washed  and  killed  bacilli  into  animals,  or  by  freezing  the  bacilli 
with  liquid  air,  and  then  crushing  them.  This  injected  into 
guinea  pigs  causes  diarrhoea,  mydriasis  and  death. 

Oxygen  Requirements. — It  is  a  facultative  aerobe. 

Cultural  Characteristics. — It  grows  upon  all  media  at  the  tem- 
perature of  the  body,  37°  C.  and  more  slowly  at  20°  C.  On  gela- 
tine plate  it  produces  at  first  small  colonies,  yellowish  and  punctate, 
which  become  whitish,  delicately  notched  and  ridged.     (Fig.  50.) 


TYPHOID   BACILLUS  151 

In  gelatine  stab  culture  it  grows  in  a  thread-like  granular  line, 
without  producing  gas.  In  neither  case  is  the  gelatine  liquefied. 
On  agar  plates  the  colonies  are  not  so  characteristic,  being  round, 
grayish- white,  and  shining.  In  milk  it  grows  well,  not  coagulating 
it  even  after  boiling,  and  only  a  very  little  acid  is  produced.  On 
acid  potato  the  growth  is  characterized  by  its  invisibility,  _  and 
this  fact  is  used  to  differentiate  it  from  other  kindred  bacteria.  The 
growth  is  only  detected  by  scratching  with  a  needle.  In  bouillon 
it  grows  uniformly,  producing  very  little  acid,  and  no  demonstrable 
amount  of  gas.  In  special  media  (Hiss's  semi-solid  media)  thread- 
like colonies  are  produced,  which  are  characteristic.  On  Eisner's 
potato  media  it  produces  small  granular,  glistening  points.  It  also 
grows  characteristically  in  Capaldi  and  the  Drigalski  and  Conradi 
media. 

Invasion  of  Body. — This  organism  generally  invades  the  body 
by  way  of  the  alimentary  tract,  in  food  and  water.  Flies  may  infect 
milk  and  other  foods.  Oysters  may  become  infected  and  cause 
disease. 

Pathogenesis. — It  is  certainly  the  cause  of  typhoid  fever.  Is 
found  in  the  stools  and  urine  of  the  patient,  and  may  be  recovered 
from  the  blood.  Also  found  in  the  spleen  and  gall  bladder.  It 
produces  well  marked  histological  changes  in  the  lymphoid  struc- 
tures, particularly  in  Peyer's  patches,  solitary  follicles,  and  other 
lymph  glands.  There  is,  according  to  Mallory,  a  massive  endothe- 
lial proliferation  in  the  lymph  glands.  This  causes  occlusion  of 
the  lymph  vessels,  and  is  followed  by  necrosis  (ulceration)  of  the 
Peyer's  patches.  The  intense  phagocytic  action  of  the  fixed  lym- 
phatic cells  in  the  glands  is  manifest  toward  the  red  blood  cells, 
which  are  devoured  in  great  numbers.  The  toxin  causes  degenera- 
tion of  other  organs,  particularly  in  the  liver.  Bacilli  are  found  in 
the  spleen  and  blood.  The  rose  colored  spots  are  found  to  be  full 
of  them.  The  disease  is  certainly  not  a  merely  localized  infection 
of  the  lymph  structures,  but  is  a  bacteraemia.  There  is  often  a 
mixed  infection  in  which  streptococcus  pyogenes  in  the  blood 


152  BACTERIA 

plays  an  active  r6le.  In  the  necrosis  of  bone  and  in  subphrenic 
abscess  the  typhoid  bacilli  may  act  as  a  pus  former.  Commonly 
it  produces  death  by  (i)  profound  toxaemia;  (2)  ulceration  of  the 
Peyer's  patches,  causing  perforation  and  peritonitis;  (3)  by  the 
destruction  of  a  blood  vessel  in  the  floor  of  an  ulcer  producing  a 
haemorrhage. 

In  animals,  as  a  rule,  typhoid  bacilli  if  injected,  produce  no  dis- 
ease, and  the  bacilli  rapidly  die.  In  chimpanzees,  however,  it  is 
possible  to  produce  typical  typhoid  lesions  and  symptoms. 

Natural  and  Acquired  Immunity. — Human  blood  serum  is 
strongly  bactericidal  toward  the  typhoid  bacillus.  Normal  gastric 
juice,  with  its  hydrochloric  acid,  destroys  the 
bacillus  when  ingested  and  this  forms  the 
natural  means  of  protection.  Immunity  follow- 
ing an  attack  of  typhoid  is  generally  of  long 
duration.  If  bacilli  do  reach  the  blood  stream 
of  an  immune  individual,  the  amboceptors 
originated  by  a  previous  infection,  together 
action.  On7h!lfof  tii^;  "^^^^  the  complement  normally  present,  effect 
field  shows  typhoid  a  solution  of  the  invading  organism.  Artifi- 
o  ?h  i  V '  hX^^'Siows  ^^^1  immunity  has  been  effected  against  typhoid 
clumping.  (Greene's  by  vaccinating  individuals  with  killed  cultures. 
Medical  Diagnosis.)  Anti-toxin  for  typhoid  has  been  prepared  by 
injecting  horses  with  killed  culture  of  typhoid  bacilli,  but  it  has 
not  proved  to  be  effective. 

Agglutinations. — One  of  the  most  important  means  of  diagnos- 
ing typhoid  fever  is  by  the  so-called  Widal  test,  really  the  Gruber  and 
Durham  agglutination  reaction.  This  consists  in  applying  the 
serum  of  the  blood  of  a  person,  supposedly  ill  with  typhoid,  to  a 
fresh  bouillon  culture  of  typhoid  bacilli.  If  the  person  has  the  dis- 
ease, and  it  has  lasted  for  five  or  more  days,  the  bacilli  are  promptly 
agglutinated  in  clumps.  Undiluted  normal  serum,  and  serum  from 
people  suffering  other  diseases,  will  bring  about  the  same  reaction  at 
times;  it  is  therefore  best  to  dilute  the  serum  with  water  1-50,  and 


TYPHOID   BACILLUS  1 53 

if  the  reaction  comes  within  an  hour  the  disease  is  considered  typhoid 
fever.  The  test  may  be  either  with  a  hanging  drop  and  examined 
microscopically,  or  macroscopically  by  adding  a  drop  of  diluted 
serum  to  fresh  bouillon  culture  of  typhoid  bacilli,  when,  if  the  case 
is  typhoid,  large  clumps  of  the  bacilli  will  form  and  drop  to  the 
bottom  of  the  tube.  Animals  immunized  against  typhoid  exhibit 
this  reaction  to  a  high  degree.  Serum  diluted  with  10,000  parts 
of  water  has  caused  the  reaction  in  less  than  one  hour's  time.  This 
reaction  with  a  known  culture  of  typhoid  bacilli  is  used  clinically 
to  identify  serum  from  a  doubtful  case  of  typhoid,  and  estabh'sh  a 
diagnosis.  On  the  other  hand,  a  known  serum  prepared  artificially 
by  immunizing  rabbits  with  bacilli  is  used  to  identify  typhoid  bacilli 
when  found  in  water,  or  elsewhere.  The  fetus  of  a  woman  suffering 
from  typhoid  contains  agglutinins  in  its  blood.  The  milk,  tears, 
and  other  body  fluids  from  an  individual  with  typhoid,  agglutinate 
typhoid  bacilli.  Serum  to  perform  the  test  may  be  obtained  by 
puncturing  the  skin,  or  by  blistering  it  and  drawing  off  the  serum, 
or  else  by  abstracting  blood  from  a  vein  with  a  hypodermic 
syringe. 

Agglutinin  appears  during  typhoid,  generally  after  the  fifth  day, 
and  persists  for  some  time  (several  years?)  after  convalescence. 

There  are  two  stages  to  the  reaction;  immediately  after  mixing 
the  serum  and  culture,  the  bacilli  will  be  seen  to  become  less  motile, 
and  then  still.  After  this  they  begin  to  huddle  together  into  clumps. 
In  complete  reaction  they  remain  immobile  and  tightly  massed. 
In  some  cases  bacteriolysis  occurs,  and  many  of  the  bacteria  are 
dissolved  in  the  serum.  It  is  still  uncertain  whether  the  reaction 
is  merely  a  phenomenon  of  infection,  or  whether  it  has  to  do  with 
immunity.  By  many  it  is  held  that  the  two  are  distinct  and  separate 
and  that  it  is  a  phenomenon  of  infection.  There  are  several  reasons 
for  thinking  so.  i.  The  bactericidal  action  of  serum  is  destroyed 
at  56°  C.  The  agglutinating  power  is  not  destroyed  at  62°  C.  2. 
A  serum  may  be  bactericidal,  but  not  agglutinative.  3.  Bacteria 
treated  with  bactericidal  sera  lose  their  virulence,  and  those  that 


154  BACTERIA 

have  been  agglutinated  do  not  do  so.  (Compare  Friedberger's 
idea  of  infection,  page  59.) 

Paratyphoid  Bacillus. — A  pathogenic  organism  producing  all 
the  clinical  symptoms  of  typhoid,  only  in  milder  form  (at  times)  has 
lately  been  discovered.  It  differs  from  the  true  bacillus  because  it 
ferments  dextrose  and  maltose  producing  gas  and  acid,  and  is  not 
agglutinated  by  the  serum  from  a  true  typhoidal  infection,  even  in 
high  dilution.  Various  varieties  differ  in  growth  upon  litmus  milk. 
In  every  other  respect  it  resembles  the  typhoid  bacillus,  and  seems 
to  occupy  a  position  betv^een  it  and  the  colon  bacillus.  Paratyphoid 
endotoxin  resists  60°  C.  from  thirty  to  sixty  minutes,  so  it  is  said. 

The  Paracolons  are  organisms  like  the  paratyphoids,  but  some- 
what closer  to  the  colon  bacillus.     (For  example,  see  page  156.) 

Blood  cultures  are  often  employed  in  large  hospitals  for  the  diag- 
nosis of  typhoid  fever.  During  the  first  week  of  the  attack  bacilli 
may  be  recovered  from  the  blood  by  withdrawing  10  c.c.  of  blood 
from  a  vein  and  mixing  it  with  500  c.c.  of  bouillon.  The  large 
amount  of  blood  is  necessary,  because  the  bacilli  are  few  in  number, 
and  the  bactericidal  action  of  the  serum  outside  the  body  is  powerful 
until  mixed  with  the  bouillon,  after  which  the  bacilli  are  able  to 
vnthstand  it.  The  bacilli  may  be  easily  isolated  from  the  blood  by 
adding  the  latter  to  some  bile  and  then  incubating  it.  From  the 
bile,  cultures  are  made  in  agar  or  in  bouillon. 

COLON  BACILLUS. 

Bacterium  Coll. 

Bacillus  coli  or  Bacillus  coli  communis. 

Colon  Bacillus. 

While  not  strictly  a  pathogenic  organism,  it  plays  such  an  impor- 
tant part  in  secondary  infection,  and  resembles  so  closely  the 
typhoid  bacillus,  that  it  will  be  described  here. 

Morphology  and  Stains. — Is  not  so  motile  as  typhoid;  has 
not  so  many  flagella;  and  is  devoid  of  spores.     It  exhibits  pleomor- 


COLON   BACILLUS  1 55 

phism;  may  grow  in  chains;  and  possesses  vacuoles  and  polar 
bodies  at  times.  Is  readily  stained  by  all  the  common  basic  stains, 
but  not  by  Gram's  method. 

Oxygen  Requirements. — It  grows  especially  well  in  oxygen. 
Without  oxygen  its  growth  is  not  so  good. 

Temperature  Requirements,  and  vital  resistance.  It  grows  well 
at  room  and  incubator  temperature.  Its  thermal  death-point  is 
about  62°  C. ;  light  and  heat  are  destructive  to  it,  and  its  resistance 
to  antiseptics  is  somewhat  better  than  that  of  typhoid  bacillus. 

Cultures. — Thrives  in  all  common  culture  media,  especially  if 
sugar  is  present.     It  is  restrained  by  excess  of  acids  produced  in 


Fig.  52. — Colon  bacillus  showing  flagella.     (KoUe  and  Wassermann.) 

culture  media.  On  gelatine  it  grows  like  the  typhoid  bacillus  (from 
which  it  is  difficult  to  differentiate,  see  page  263)  in  whitish  raised 
colonies  that  do  not  liquefy  the  media.  Sometimes  the  growth  is 
thin  and  iridescent,  and  exhibits  bizarie  shapes — tadpole-like  and 
lobulated.  Typhoid  colonies  show  deep  furrow-like  r'dges  under 
the  microscope.  In  the  special  semi-solid  media  of  Hiss,  the  typhoid 
produces  uniform  cloudiness,  with  thread-like  colonies.  The 
colon  does  not  so  quickly  cause  this  cloudiness,  and  forms  gas 


156  BACTERIA 

bubbles.  On  agar  plates  surface  colonies  are  like  typhoid,  only 
they  are  thicker  and  moister.  If  litmus  is  added  to  this  medium,  a 
red  zone  forms  about  the  colonies,  due  to  the  presence  of  lactic  acid. 
In  agar  tubes  the  growth  is  more  luxuriant  and  resembles  typhoid. 
In  litmus  bouillon  it  rapidly  reddens  the  litmus,  clouds  the  medium, 
and  deposits  a  slimy  sediment.  In  milk  it  always  produces  coagu- 
lation. On  potato  it  grows  more  rapidly  and  luxuriantly  than 
typhoid,  at  first  yellowish-white,  which  later  changes  to  yellowish- 
brown.     It  is  slimy. 

Chemical  Activities. — Produces  color  on  potato  only.  Sugars 
are  fermented  with  the  production  of  H,  CO2  and  some  N.  Some 
varieties  ferment  cane  sugar.  Produces  lactic,  acetic  and  formic 
acids,  also  indol  abundantly,  and  H2  S.  It  decomposes  urea.  There 
are  a  great  many  varities  of  colon  bacilli  having  very  different 
chemical  activities. 

Habitat. — Found  always  in  the  intestinal  contents  of  most  ani- 
mals and  man.  Also  in  streams  and  rivers  that  run  through  farm 
lands  and  by  towns.  While  it  is  difficult  to  find  typhoid  bacilli  in 
drinking  water,  the  colon  bacilli  are  easily  found.  If  in  abundance, 
it  indicates  great  fecal  pollution.  In  milk  it  is  often  found,  where 
it  plays  an  important  part  in  souring. 

Pathogenesis. — It  is  pathogenic  to  rabbits  and  guinea  pigs, 
causing  peritonitis  if  injected  into  the  peritoneal  cavity.  In  man 
it  plays  rather  a  subordinate  pathogenic  role,  but  it  has  been  found 
the  causal  agent  of  some  cases  of  suppurative  appendicitis,  peri- 
tonitis, and  cystitis.  It  may  attack  the  lungs  and  meninges  of 
feeble  children,  and  cause  death  by  setting  up  a  pneumonia  or 
meningitis.  During  the  agonal  period  in  wasting  diseases  it  may 
cause  terminal  infection  and  death.  Colon  bacilli  encysted  in  the 
liver  and  kidney  have  been  found  by  Adami  in  cirrhosis  of  these 
organs,  and  it  is  believed  by  him  to  be  partly  the  cause  of  these 
diseases;  chronic  infections  of  the  rectum  are  due  to  this  organism. 

Agglutination. — Animals  immunized  against  colon  bacilli  by 
repeated  injections,  exhibit  agglutinins  in  their  blood. 


DYSENTERY  BACILLUS  1 57 

The  differentiation  of  the  typhoid  from  the  colon  bacillus  is  largely 
accomplished  by  noting  the  chemical  reactions  of  both  organisms 
in  culture  media.     The  chief  differences  are: 

1.  The  typhoid  bacillus  has  more  flagella  than  the  colon,  and  is 
much  more  motile. 

2.  On  gelatine  culture  plates,  the  typhoid  colonies  develop  more 
slowly  than  the  colon,  and  are  much  more  delicate  and  transparent. 
If  litmus  is  present  the  colon  colonies  are  red,  the  typhoid  bluish. 

3.  In  media  containing  dextrose,  or  lactose,  gas  is  produced  by 
the  colon,  but  not  by  typhoid. 

4.  In  peptone  solution  the  colon  produces  indol,  while  the  typhoid 
does  ngt. 

5.  Milk  is  coagulated  by  the  colon,  but  not  by  the  typhoid. 

6.  On  potatoes  colon  grows  much  more  luxuriantly  than  typhoid. 

7.  Typhoid  reddens  neutral  red;  colon  changes  it  to  bright  yellow. 

8.  The  most  important  test  is  the  agglutinative  one.  Typhoid  is 
clumped  by  anti-typhoid  sera,  highly  diluted,  while  the  colon  is  not. 

No  anti-sera  of  value  have  been  found  for  colon  bacillus  infection, 
but  bacterins  have  been  used  with  much  benefit. 

DYSENTERY  BACILLUS. 

Bacterium  Dysenteriae. 

Dysentery  Bacillus  of  SJiiga  and  Flexner. 

Supposed  cause  of  one  form  of  tropical  dysentery.  The  group 
to  which  this  belongs  comprises  many  closely  related  varieties  some 
of  which  are  thought  to  be  the  cause  of  infant  diarrhoea  in  this 
country.  There  are  various  strains  of  this  organism,  the  differentia- 
tion of  which  depend  upon  their  chemical  activities,  fermentation 
of  various  carbohydrates  being  the  most  important,  and  agglutina- 
tive properties  with  different  sera. 

Morphology  and  Stains. — The  organism  is,  in  many  respects, 
similar  to  the  typhoid  bacillus,  but  is  plumper.  It  is  said  to  be 
flagellated,  has  no.  spores,  and  exhibits  pleomorphism.  It  stains 
well  with  the  common  aniline  dyes,  but  not  by  Gram's  method. 


158  BACTERIA 

Vital  Properties. — It  is  killed  by  i  percent  carbolic  solution 
in  thirty  minutes.  Lives  for  twelve  to  seventeen  days  v^^hen  dried. 
Direct  sunlight  kills  it  in  thirty  minutes.  Its  thermal  death-point 
is  58°  C.  in  thirty  minutes.  It  is  a  facultative  aerobe;  grov^^s  at 
ordinary  temperature,  but  better  at  37°  C. 

Cultures. — Grows  on  all  the  common  culture  media,  but  more 
slowly  than  the  colon  bacilli.  Gelatine  cultures  resemble  typhoid. 
The  growth  in  this  media  (which  it  does  not  liquefy)  produces  no 
pellicle,  but  a  sediment.  Indol  is  not  produced,  and  milk  is  first 
mildly  acid  and  then  faintly  alkaline,  though  not  coagulated.  On 
potato  it  grows  sparingly,  often  turning  it  brown.  The  Shiga 
type  ferments  glucose,  but  no  other  sugar.  The  Flexner  type 
ferments  glucose,  dextrine,  and  mannite,  but  not  lactose.  The 
latter  type  produces  more  acid  than  the  former,  and  both  are  best 
agglutinated  with  their  corresponding  serums. 

Habitat. — In  living  bodies  the  organism  is  found  solely  in  mucous 
discharges  from  the  bowels.  In  the  dead  it  is  found  in  the  lymph 
glands.  If  it  reaches  the  circulation,  it  appears  to  be  rapidly 
destroyed  by  the  blood.  It  has  been  discovered,  however,  in  the 
body  of  a  foetus  delivered  from  a  woman  with  the  disease.  The 
organism  must  have  passed  the  placenta  of  the  mother.  The 
disease  is  spread  by  water,  and  it  may  become  epidemic  in  large 
institutions. 

Pathogenesis. — The  typical  lesions  caused  by  the  organism  vary 
from  a  mere  hyperaemia  to  a  superficial  necrosis  of  the  lymphoid 
structures,  which  may  be  extensive.  Peyer's  patches  are  slightly 
swollen  but  not  ulcerated.  The  descending  colon  and  sigmoid 
are  oftenest  attacked.  The  necrotic  masses  separate,  leaving 
shallow  ulcers.  The  lymph  structures  are  engorged  with  polynuclear 
leucocytes.  No  marked  lesion  is  found  in  the  spleen.  The  liver 
and  kidneys  often  undergo  marked  parenchymatous  degeneration. 
The  bacilli  being  possessed  of  a  powerful  endo-toxin,  so  that  dead 
cultures,  if  injected  under  the  skin  cause  marked  local  and  general 
reactions.     Like  the  pyocyaneus  bacillus,  this  organism  undergoes 


DYSETERY  BACILLUS  159 

auto-digestion  in  bouillon,  which  leaves  the  latter  highly  toxic  owing 
to  the  liberation  of  the  toxins.  Laboratory  animals  quickly  suc- 
cumb to  injection  of  this  organism,  injection  producing  a  marked 
reaction  in  the  colon,  a  phenomenon  suggesting  that  there  is  a 
predilection  for  the  organ  and  that  the  body  uses  it  as  an  excretory 
organ  for  the  poison.  Dysentery  cannot  be  induced  in  animals  by 
feeding  cultures.  Poorly  nourished  subjects  are  easily  infected 
and  quickly  die.  Digestive  disorders  favor  infection.  Death  may 
be  due  to  toxaemia  or  exhaustion.  As  a  causal  agent  in  the  produc- 
tion of  summer  diarrhoeas  of  children,  the  dysentery  bacillus  plays 
a  part.  It  has  been  isolated  from  the  stools  of  infants,  with  this 
disease,  and  their  sera  have  been  found  to  agglutinate  the  bacilli. 
Nevertheless  it  is  known  that  other  bacteria  (streptococci,  etc.) 
cause  this  disease,  and  Weaver  found  that  ''clinically  twenty-four 
of  our  ninety-seven  cases  of  ileocolitis  in  which  dysentery  bacilli 
were  discovered  did  not  differ  from  cases  in  which  dysentery  bacilli 
were  not  found. 

Immunity. — The  sera  from  convalescents  from  dysentery  shows 
a  strong  bactericidal  action.  Anti-bodies  are  developed  by  infection 
and  by  artificial  inoculation  with  killed  cultures.  Kruse  obtained  a 
serum  from  horses  which  strongly  protected  a  guinea  pig  against  a 
lethal  injection  of  bacilli.  The  protective  property  of  the  serum  is 
due  to  its  bactericidal  action.  Here  the  amboceptors  act,  but  only 
in  the  presence  of  a  complement.  Anti-toxic  sera  protected  against 
bacteria;  and  an  anti-bacterial  serum  protected  against  toxin, 
according  to  Rosenthal. 

Vaccination. — Shiga  tried  to  induce  (i)  passive  and  (2)  active 
immunity  in  many  individuals  by  injecting  both  anti-toxic  serum 
and  bacteria  into  them.  This  was  not  followed  by  a  lowered 
number  of  infections,  but  by  a  lowered  mortality.  A  serum  may 
be  produced  by  injecting  horses  with  several  dysentery  strains, 
called  a  polyvalent  anti-serum.  This  has  good  therapeutic  effects 
but  does  not  immunize  prophylactically. 

Agglutination. — The    serum   from    a   patient    suffering   from 


l6o  BACTERIA 

either  dysentery  or  summer  diarrhoea,  will,  after  about  a  week's 
illness,  agglutinate  bacilli.  This  property  is  not  always  present, 
and  its  absence  does  not  exclude  the  possibility  of  infection.  In 
performing  the  reaction,  both  Shiga's  and  Flexner's  type  of  organ- 
ism should  be  used.  These  types  probably  bear  the  same  relation 
to  each  other  that  typhoid  and  paratyphoid  do. 

GARTNER'S  BACILLUS. 

Bacillus  Enteritidis. 

Bacillus  of  Gartner. 

The  cause  of  one  form  of  meat  poisoning,  and  closely  allied  to 
the  paratyphoid  bacillus  in  its  morphological  characteristics. 
It  gives  a  classical  picture  of  the  type  "paracolon." 

Morphology  and  Stains. — This  organism  is  a  short  plump 
ovoid;  is  motile;  has  about  eight  flagella;  does  not  form  spores; 
and  stains  well  with  all  the  common  aniline  dyes,  but  not  with 
Gram's  method.     It  forms  a  slight  capsule. 

Vital  Resistance. — It  is  a  facultative  anaerobe.  It  is  destroyed 
by  means  outlined  for  the  colon  bacillus  when  in  culture.  In 
meat  it  must  be  subjected  to  prolonged  heating. 

Cultures. — Grows  on  all  the  common  culture  media.  In 
bouillon  thrives  well,  producing  gas  in  media  containing  dextrose. 
It  ferments  without  gas  production  lactose,  galactose,  maltose,  and 
cane  sugar.  Does  not  produce  indol,  which  distinguishes  it  from 
the  colon  bacillus,  to  which  it  is  closely  allied.  In  milk  it  reduces 
litmus  and  coagulates  the  casein  in  a  few  days.  On  potato  it  grows 
well,  producing  a  yellowish  shining  layer.  On  gelatine  it  multiplies 
without  liquefying  the  medium.  Superficial  colonies  in  plates  are 
pale  and  gray,  deep  colonies  yellow  and  spherical. 

Chemical  Activities. — Acid,  gas  and  a  powerful  heat-resisting 
toxin  which  is  soluble,  are  found.  Infected  meat  contains  this 
toxin,  which  is  not  destroyed  by  cooking. 

Pathogenesis. — It  is  pathogenic  for  man,  horses,  cattle,  and 


PYOCYANEUS   BACILLUS  l6l 

laboratory  animals.  Neither  the  bacilli  or  the  toxin  they  elaborate 
are  destroyed  by  heat.  Flesh  is  infected  before  death,  after  which, 
both  the  bacilli  and  toxin  increase.  Mischief  follows  the  partaking 
(usually  in  the  form  of  sausages,  etc.)  of  this  meat,  causing,  in  men, 
violent  nausea  and  diarrhoea,  skin  eruption,  and  in  severe  cases, 
pneumonia,  nephritis,  collapse  and  death.  Mortality  is  from  2 
percent  to  15  percent.  The  post  mortem  findings  are  not  specific. 
There  may  be  evidence  of  an  enteritis  with  swollen  lymph  follicles, 
and  an  enlarged  spleen. 

Agglutination. — The  blood  of  infected  individuals  may  agglu- 
tinate bacilli.  A  dilution  of  such  blood  with  8,000  parts  of  water 
has  produced  the  reaction. 

No  anti-serum  or  bacterin  treatment  is  as  yet  possible. 

PYOCYANEUS  BACILLUS. 

Bacterium  Pyocyaneus. 

Bacillus  Pyocyaneus.     (Fig.  53.) 

Bacillus  of  Blue  Pus.     Also  called  Pseudomonas  pyocyanea. 

An  organism  of  minor  importance  as  a  pathogenic  agent,  that  is 
often  met  with  in  groin  or  axilla. 

Morphology  and  Stains. — Slender  rods,  often  growing  into 
thread-like  forms.  Exhibits  pleomorphism.  Sometimes  is  rounded 
and  cocci-like,  is  motile,  has  a  polar  flagellum,  and  no  spores. 
Stains  with  all  the  basic  aniline  dyes,  but  not  with  Gram's  method. 

Oxygen  Requirements. — Usually  a  strict  aerobe. 

Cultures. — Grows  on  all  the  common  culture  media  luxuriantly, 
at  room  and  incubator  temperatures.  It  elaborates  two  pigments, 
a  water-soluble  greenish  bacteriofluorescein,  and  a  chloroform- 
soluble  pigment,  a  beautiful  blue  in  color,  called  pyocyanin.  On 
gelatine  plates  it  produces  yellowish-white  to  greenish-yellow 
colonies  which  liquefy  the  gelatine,  causing  crater-like  excavations 
about  the  colonies.  Gelatine  stab  cultures  rapidly  liquefy  along 
the  line  of  inoculation,  coloring  the  gelatine  greenish-blue,  and  a 


1 62  BACTERIA 

white  crumbly  deposit  forms  in  the  bottom  of  the  stab.  On  agar 
plates  it  produces  yellowish-white  colonies,  surrounded  by  a  zone 
of  bluish-green  fluorescence.  It  grows  luxuriantly.  In  agar  tubes 
it  multiplies  rapidly,  spreading  over  the  medium,  with  wavy  thick- 
ened edges.  The  agar  quickly  turns  a  dark  greenish-blue,  and 
in  old  cultures  the  growth  changes  from  yellow  to  greenish-blue. 


Fig.  53. — Bacillus  pyocyaneus.     (Kolle  and  Wassermann.) 

In  bouillon  it  is  very  dense  and  yellowish-green;  a  pellicle  forms 
on  the  surface,  and  a  sediment  is  deposited.  In  old  bouillon  cul- 
tures the  bacilli  undergo  autolysis  and  disappear.  In  milk  the 
growth  is  luxuriant,  the  casein  is  coagulated,  and  the  clot  is  ulti- 
mately digested.  The  reaction  is  alkaline.  On  potato  it  varies 
in  luxuriance,  often  being  slightly  elevated,  yellowish,  turning  to 
green.  The  variance  in  growth  is  due  to  the  kind  of  potato  used. 
Drying  kills  the  organism  speedily;  four  hours  in  sunlight  also  de- 
stroys it. 

Chemical  Activities. — No  gas  is  generated.  Besides  the  pig- 
ments (already  specified)  ammonia  is  produced,  also  a  peculiar 
enzyme  called  pyocyanase  by  Emmerich  and  Lowe,  which  not  only 
digests  gelatine  and  milk-curd,  but  its  own  and  other  bacterial  cells 


BACILLUS    OF   SOFT   CHANCRE  1 63 

themselves.  Old  cultures  are  poisonous ;  a  haemolysin  is  produced — 
an  endo-toxin,  and  a  soluble  toxin.  Against  the  endo-toxin  and  the 
soluble  toxin  it  is  possible  to  prepare  an  anti-serum.  This  may  pro- 
tect laboratory  animals.  The  last  named  toxin  stands  a  tempera- 
ture of  100°  C. 

Pathogenesis. — Has  been  found  a  sole  cause  of  meningitis  and 
vegetative  endocarditis  in  man;  is  a  pyogenic  organism;  can  cause 
suppuration  anywhere  in  the  body;  produces  blue  pus;  is  pathogenic 
to  guinea  pigs;  and  its  virulence  can  be  raised  by  passing  it  through 
a  series  of  animals. 

Agglutination. — The  serum  of  infected  and  immunized  animals 
both  in  moderate  dilution  causes  agglutination  of  bacilli.  It  is 
possible  to  use  bacterins  of  this  germ.  Bactericidal  substances 
develop  by  the  use  of  killed  cultures. 

BACILLUS  OF  SOFT  CHANCRE. 

Bacterium  Ulceris  Chancrosi.     (Ducrey.) 

Streptohacillus  of  Soft  Chancre. 

Morphology  and  Stains. — A  small  thin  bacterium  .5//  broad, 
1.5/^  long,  growing  in  chains  with  polar  staining,  which  can  be 
demonstrated  in  sections  of  chancres  without  much  difficulty. 

This  organism  does  not  stain  by  Gram's  method,  but  by  Loffler's 
it  is  stained  with  ease. 

Cultures  are  hard  to  make.  It  grows  best  in  serum  agar,  and 
blood  agar  in  faint  colonies  that  are  not  very  characteristic.  In 
condensation  water  of  agar  it  grows  feebly. 

In  sections  and  in  pus  the  organism  is  frequently  found  in  the 
interior  of  leucocytes. 

By  aspirating  pus  from  buboes  and  planting  it  in  blood  agar 
cultures  may  be  obtained. 

Pathogenesis. — From  an  old  culture  of  over  ten  generations 
typical  ulcerations  were  produced  in  man.  The  organism  is  feeble 
and  quickly  dies  in  culture  media  or  in  contact  with  mild  antiseptics. 


164  BACTERIA 

ANTHRAX  BACILLUS. 

Bacillus  Anthracis. 

Anthrax  Bacillus  of  Koch.     (Fig.  54.) 

Practically  the  first  pathogenic  organism  to  be  isolated.  This 
was  accomplished  by  Dr.  Robert  Koch.  It  is  the  cause  of  a  wide- 
spread malignant  disease,  variously  called  Anthrax,  Charbon,  or 
Splenic  Fever.  Animals  and  man  are  infected  by  it,  and  its  action 
is  often  rapidly  fatal. 

Morphology  and  Stains. — In  animal  tissues  this  organism  ap- 
pears as  a  large  rod  3-10//  long,  and  1-1.2 }jl  wide.  Is  often  in  pairs 
or  chains.  In  fresh  specimens  the  ends  of  the  rods  are  rounded; 
when  older,  the  ends  become  square  or  con- 
cave. Often  they  have  faint  capsule  sur- 
rounding them.  In  culture  media  they  ex- 
hibit spores  and  grow  in  long  threads,  these 
threads  form  long  spirally  twisted  masses,  like 
locks  of  wavy  hair.  No  flagella  are  formed, 
and  the  organism  is  not  motile.     In  old  cul- 

Ko  ^^ik'^'T!   ^1  ^^A    tures,  bizarre  involution  forms  are  found.     It 
bacilli     in     blood.  ' 

(Greene's   Medical  stains  well  with  all  the  common  basic  dyes 

Diagnosis.)  ^^^  ^^  ^^^^,^  method. 

Oxygen  Requirements. — Is  a  facultative  anaerobe,  but  grows 
much  better  in  the  presence  of  oxygen.  If  oxygen  is  excluded,  no 
liquefaction  occurs. 

Temperature. — Grows  between  14°  C.  and  45°  C;  best  at  37°  C. 
Spores  are  formed,  if  oxygen  is  abundant,  above  12°  C.  Sporu- 
lationis  more  rapid  at  37°  C.  Spores  withstand  high  temperature 
(dry)  for  a  long  time,  100°  C.  for  one  hour.  The  bacillus  itself  is 
killed  at  70°  C,  moist  heat,  in  one  minute.  The  thermal  death- 
point  may  be  put  down  for  the  organism,  at  100°  C.  moist,  for 
five  minutes. 

Vital  Resistance. — Highly  resistant  to  chemicals,  light  and  dry- 
ing.    Spores  resist  5  percent  carbolic  solution  for  days  (Esmarch), 


ANTHRAX  BACILLUS  1 65 

and  I  percent  corrosive  sublimate  (aqueous)  for  three  days.  They 
also  resist  formaldehyde  and  sulphur  for  a  long  time,  and  withstand 
light.  A  2  percent  fresh  solution  of  H^Oa  kills  spores  in  three 
hours.  Three  and  one-half  hours'  exposure  to  bright  sunlight  killed 
the  spores  if  oxygen  was  not  excluded.     (Dieudonnd.)     (Fig.  55.) 


Fig.  55. — Anthrax  bacilli  growing  in  a  chain  and  exhibiting  spores.     (KoUe  and 

Wassermann.) 

Sporulation  Phenomena. — At  12°  C.  spores  are  formed  if 
oxygen  is  present.  The  most  favorable  temperature  for  sporula- 
tion is  that  of  the  body  (37°  C).  Spores  are  never  found  in  the 
bodies  of  living  or  dead  animals  if  they  remain  unopened,  and 
oxygen  is  excluded.  If  bacilli  are  cultivated  at  42°  C.  for  a  long 
time  and  frequently  reinoculated,  on  fresh  media,  the  ability  to 
form  spores  is  lost  even  if  grown  again  at  30°  C.  (Phisalix).  If 
cultivated  upon  media  containing  carbolic  acid  and  hydrochloric 
acid,  the  ability  to  sporulate  may  be  lost. 

Chemical  Activities. — Acetic  acid  is  formed,  as  is  HgS.  Lique- 
fying, milk  coagulating,  and  milk  digesting  enzymes  are  formed. 
Toxins  have  not  been  isolated,  but  may  be  produced. 

Habitat. — Only  found  where  infected  animals,  hides,  and  hair 


I 66  BACTERIA 

have  been.  Fields,  hay,  bristles,  hides,  manure,  etc.,  have  been 
found  to  contain  bacilli.  Drinking  water  may  be  polluted  by  tan- 
neries and  the  bodies  of  dead  animals.  Meadows  and  fields  may 
be  contaminated  for  years.  From  the  buried  bodies  of  infected 
animals  anthrax  spores  may  be  brought  to  the  top  of  the  soil  by 
earth-worms. 

Cultures. — Grows  exceedingly  well  on  all  culture  media  in  the 
air.  On  gelatine  it  grows  in  whitish  round  colonies,  rapidly  sink- 
ing into  the  gelatine,  due  to  the  liquefaction.  The  liquid  medium  is 
turbid.  The  interior  of  the  colony  is  crumbly.  When  magnified, 
the  colonies  seem  to  be  made  up  of  tangled  waving  bundles,  like 
locks  of  hair,  especially  about  the  periphery.  In  gelatine  stab 
cultures  the  growth  is  luxuriant  and  rapid;  the'medium  is  liquefied 
more  rapidly  at  the  top,  and  finally  a  crater  is  formed;  before  this 
appears,  lateral  hair-Hke  outgrowths  are  ^een  in  the  gelatine.  At 
the  bottom  of  the  crater  a  white  crumbly  mass  is  formed,  but  no 
pellicle.  On  agar  plates,  small  whitish  colonies  develop  which 
are  elevated  and  round.  When  magnified,  wavy  hair-like  growths 
appear  on  the  edge,  caused  by  many  twisted  parallel  chains  of 
bacilH.     (Fig.  56.) 

In  agar  stab,  the  growth  ismore  luxuriant  near  the  top;  lateral 
filamentous  branches  are  seen  along  the  stab  line.  In  agar  streak 
the  colonies  are  abundant,  thick  and  fatty;  have  tangled  edges, 
and  the  water  of  condensation  is  cloudy.  In  bouillon,  it  forms 
homogeneous  flocculi,  which  precipitate,  leaving  the  bouillon  clear. 
A  fragile  pellicle  is  formed.  In  milk,  it  multiplies  rapidly,  the 
proteids  are  coagulated,  generally  rendered  acid,  and  later  the  coagu- 
lum  is  dissolved.  Potato  cultures  are  likewise  luxuriant.  The 
growth  is  elevated,  dull  in  lustre,  and  the  outline  is  wavy. 

Pathogenesis. — The  anthrax  bacillus  increases  so  rapidly,  and 
so  luxuriantly,  that  it  has  been  supposed  to  cause  death  merely  by 
mechanically  overwhelming  the  animal:  absorbing  nutriment  and 
oxygen,  and  blocking  capillaries.  Its  action  is  certainly  not  purely 
toxic,  as  it  causes,  not  a  toxaemia,  but  a  bacteraemia.     It  is  especially 


ANTHRAX   BACILLUS  1 67 

virulent  for  man,  sheep,  cattle,  goats,  rabbits,  guinea  pigs,  mules, 
and  horses.  Rats  rarely  succumb.  Pigeons,  chickens,  and  dogs 
are  immune.  If  frogs  are  kept  at  a  temperature  of  30°  C.  they 
become  susceptible  to  infection.  At  their  normal  temperature  they 
are  immune.i  The  disease  produced  by  this  organism  is  known 
variously  in  different  countries  as  Anthrax,  Splenic  fever.  Wool- 
sorter's  disease.  Malignant  pustule,  and  Qiarbon.  It  frequently 
devastates  vast  herds  of  sheep,  cattle,  and  goats,  and  is  often  a 
pestilence  in  European  countries,  China,  and  South  America.     It 


Fig.  56. — Anthrax  bacilli.     Cover-glass  has  been  pressed  on  a  colony  and  then 
fixed  and  stained.     (KoUe  and  Wassermann.) 

appears  sporadically  in  the  United  States.  Its  origin  in  this  country 
can  usually  be  traced  to  infection  from  hides  or  hair  imported  from 
abroad.  In  man  it  is  frequently  fatal.  The  infection  is  first 
manifest  as  a  small  carbuncle  or  pustule,  from  this,  rapid  general 
infection,  as  a  rule,  ensues.  In  man  and  animals  anthrax  bacilli 
may  be  transmitted  from  mother  to  foetus  via  the  placenta.  The 
organism  is  found  in  enormous  numbers  in  infected  bodies,  invest- 
ing all  the  organs  and  the  blood.  Pus  is  produced,  and  tissues  are 
degenerated.  Infection  is  accompanied  by  a  high  leucocytosis  and 
fever.     There  is  often  congestion  of  the  lungs;  also  an  intense  fria- 


l68  BACTERIA 

bility  of  the  splenic  pulp,  and  all  the  glands  of  the  body  become 
enlarged,  and,  at  times,  many  of  them  suppurate.  In  wool-sorter's 
disease,  the  bacilli  are  inhaled,  and  lung  lesions  result. 

Immunity. — It  is  possible  to  immunize  animals  against  infection 
with  anthrax  by  means  of  vaccines.  By  this  means  the  lives  of 
many  thousands  of  domestic  animals  have  been  saved.  The  vac- 
cines are  made  by  growing  the  bacillus  at  42°  C.  for  various  lengths 
of  time  to  attenuate  them.  It  is  possible  but  impracticable  to 
produce  an  anthrax  anti-toxin. 

TETANUS  BACILLUS. 

Bacillus  Tetani. 

Tetanus  Bacillus.     (Fig.  57.) 
Lockjaw  Bacillus. 
First  seen  by  Nicolaier,  and  isolated  in  pure  culture  by  Kitasato. 


Fig.  57. — Tetanus  bacilli  showing  end  spores.     (KoUe  and  Wassermann.) 

Morphology  and  Stains. — Rod-shaped.  Varying  from  1.2 jj.  in 
length,  to  very  long  threads  of  20  to  40/z.  Sometimes  grow  in 
chains;  frequently  appear  like  short  drum-sticks  with  a  spore  at  one 


TETANUS   BACILLUS  1 69 

end,  which  is  either  round  or  oval.  At  times,  the  bacilli  in  chains 
sporulate.  The  organism  is  motile;  possesses  numerous  flagella 
(from  fifty  to  a  hundred)  peritrichously  arranged;  stains  well  with 
all  the  common  basic  aniline  dyes,  and  retains  the  color  in  Gram's 
method.     (Fig.  58.) 

Oxygen  Requirements. — Strictly  anaerobic  when  freshly  iso- 
lated from  earth  or  wounds,  but,  after  long  cultivation  on  culture 
media,  it  becomes  more  tolerant  to  small  amounts  of  oxygen. 

Temperature. — Grows  best  at  37°  C.     Below  14°  C.  not  at  all. 

Vital  Resistance. — Spores  resist  80°  C.  for  an  hour.  This  fact 
enabled  Kitasato  to  kill  all  other  organisms,  except  their  spores,  in 
pus.  Six  days'  exposure  to  direct  sunlight  is  needed  to  kill  the 
spores.  The  thermal  death-point  is  best  considered  as  100°  C. 
for  I  hour.  They  are  killed  in  2  hours  by  5  percent  phenol  +.5 
percent   HCl  and  in  30  minutes  by  i-iooo  HgCl2  +  .5  percent  HCl. 

Chemical  Activities. — Ferments  sugar;  produces  gas,  indol, 
alkali,  and  HgS.  which  gives  to  the  culture  an  odor  of  burnt  garlic  or 
onion;  marsh  gas,  CO2,  and  nitrogen  are  produced.  Gelatine  is 
liquefied.  The  most  important  product  of  growth  is  the  highly 
poisonous  complex  toxin,  which  is  made  up  of  tetanolysin,  and 
tetanospasmin;  the  latter  has  a  great  affinity  for  nerve  tissues.  This 
toxin  is  soluble  in  water,  and  can  be  separated  from  it  by  means  of 
ammonia  sulphate. 

Habitat. — Is  found  in  garden  soil,  hay,  manure,  and  dust. 
Has  been  found  in  cobwebs,  on  weapons,  in  cartridges,  and  in  the 
feces  of  man  and  of  animals.  It  is  said  to  have  been  found  in  the 
spinal  cord  of  a  man  who  did  not  die  of  tetanus.  It  has  also  been 
isolated  from  bronchi  in  a  case  of  rheumatic  tetanus  in  which  there 
was  no  lesion  in  the  body  (Carbon  and  Perrors).  In  disease 
it  is  found  in  the  infected  wound,  generally  in  a  deeply  punctured 
one,  which  is  usually  purulent  and  contains  but  few  bacilli.  Puer- 
peral tetanus,  and  tetanus  of  the  new-born,  are  but  varieties  of  the 
disease,  dependent  upon  the  site  of  infection,  whether  of  the  pla- 
centa or  umbilical  cord.     Tetanus  sometimes  occurs  spontaneously, 


lyo  BACTERIA 

without  a  sign  of  injury  anywhere.  Sheep  and  goats  are  suscep- 
tible to  infection,  so  are  guinea  pigs  and  rabbits.  Horses  are 
peculiarly  susceptible.  Soil,  or  manure,  getting  into  wounds,  is 
often  a  cause  of  tetanus.  Cow-dung  poultices,  mud  dressings,  or 
cobweb  applications  to  stop  haemorrhages, 
have  also  caused  the  disease.  Tetanus 
following  vaccination  is  often  due  to  infected 
virus,  the  latter  becoming  infected  from  the 
feces  of  the  vaccine-producing  cows  but  more 
commonly  is  due  to  dirt  getting  into  vacci- 
nation wounds. 

baSS-  shlti^r'pS-  Cultures.-This  organism  is  difficult  to 
trichous  flagella.  (Kolle  grow,  and  always  requires  an  atmosphere  of 
and  Wassermann.)  hydrogen. 

On  gelatine  plates,  the  colonies  appear  first  as  minute  white 
specks,  which  slowly  liquefy  the  medium.  As  it  grows,  hair-like 
threads  branch  out  into  the  medium,  and  the  colony  resembles  the 
periphery  of  a  chestnut  burr;  later,  the  white  appearance  changes  to 
yellow.  In  gelatine  stab  the  growth  is,  at  first,  whitish  along  the 
hne  of  the  needle,  eventually  the  gelatine  becomes  hquid,  and  a 
bubble  of  gas,  partly  filled  with  whitish-cloudy  Hquid  gelatine, 
appears.  On  agar  plates  the  colonies  are  ragged,  and  are  sur- 
rounded by  delicate  out-spreading  filaments.  In  deep  stab  culture, 
down  in  the  agar  and  remote  from  the  top,  a  spreading  tree-like 
form  appears,  with  spike-like  growths  in  the  agar.  Blood  serum 
is  sometimes  liquefied.  Bouillon  is  uniformly  clouded,  gas  is 
generated  if  sugar  is  present,  and  toxin  is  produced.  Milk  is, 
generally,  not  coagulated. 

All  cultures  of  tetanus  must  be  grown  under  an  atmosphere  of 
hydrogen  in  media,  from  which  all  free  oxygen  has  been  driven 
by  boiling,  or  else  abstracted  by  a  mixture  of  pyrogallic  acid  and 
sodium  hydrate.  It  is  possible  to  cultivate  the  organism  under 
mica  covering,  or  paraffine  poured  upon  freshly  boiled  media.  If 
sterile  glass  tubing  is  filled  with  agar  or  gelatine,  and  inoculated 


TETANUS   BACILLUS  171 

with  tetanus  bacilli,  then  sealed,  colonies  will  develop,  as  perfect 
anaerobic  conditions  are  thus  obtained.  Often  the  organism  grows 
best  in  the  presence  of  saprophytic  ones.  Strongly  pathogenic 
organisms  do  not  grow  well  in  culture  media,  while  comparatively 
non-virulent  ones  grow  very  well. 

Pathogenesis. — Tetanus  may  follow  any  wound,  no  matter  how 
insignificant,  though  deeply  punctured  ones,  caused  by  nails  or 
splinters,  are  more  often  followed  by  tetanus  infection,  especially 
if  the  puncture  is  sealed  by  blood  clots  or  pus,  and  so  creating  an 
anaerobic  condition  necessary  for  growth.  If  the  wound  is  on  the 
face  or  hand,  tetanus  symptoms  more  quickly  supervene,  while  if 
the  wound  is  on  the  foot,  these  are  apt  to  be  delayed.  The  sooner 
the  symptoms  appear  after  the  reception  of  the  injury,  the  more 
likely  will  the  disease  be  virulent  and  fatal.  If  spores  are  washed 
free  from  toxin,  according  to  Viallard  and  Rouget,  and  then  injected 
into  a  susceptible  animal,  they  do  not  cause  tetanus,  but  are  taken 
up  by  the  phagocytes.  In  other  words,  the  rods  not  the  spores 
produce  toxin.  Necrotic  tissue  in  wounds  favors  infection  with 
tetanus,  since  it  helps  to  fulfil  anaerobic  conditions,  and  in  some 
way  hinders  phagocytosis.  Aerobic  bacteria  favor  tetanus  infec- 
tion by  absorbing  the  free  oxygen  which  prevents  the  growth  of 
tetanus  organisms.  Free  oxygen  never  kills  the  organism  or  its 
spores,  but  merely  prevents  their  development.  Wounds  that  have, 
apparently  healed,  may  be  the  cause  of  tetanus.  The  toxin  is 
produced  rapidly  in  wounds,  or  what  is  more  likely,  some  is  intro- 
duced with  the  bacilli  and  other  dirt.  Kitasato  found,  in  the  case 
of  mice,  that  if  bacilli  were  introduced  in  the  skin,  near  the  tail,  and 
in  an  hour  the  whole  area  was  excised,  and  the  wound  cauterized, 
fatal  tetanus  nevertheless  supervened. 

Rheumatic  tetanus  follows  pulmonary  infection.  As  related  in 
the  chapter  on  toxins,  the  mode  of  disease  production  is  as  follows: — 
The  toxin  is  conveyed  from  the  wound  by  means  of  the  motor  nerves 
to  the  central  nervous  system  affecting  the  motor  elements.  It 
causes  microscopic  degeneration  of  the  fibers  and  cells  of  the  motor 


172  BACTERIA 

apparatus.  Death  is  caused  either  by  a  spasm  of  the  glottis  or 
diaphragm,  or  by  cardiac  failure  and  exhaustion.  A  local  mani- 
festation merely  affecting  certain  groups  of  muscles  may  occur. 
Laking  of  the  blood  by  tetanolysin  found  in  the  bodies  dead  from 
tetanus  is  a  well  known  phenomenon.  In  fatal  cases,  toxin  may 
be  demonstrated  in  the  bladder  by  injecting  the  urine  into  mice, 
causing  in  them  tetanic  symptoms.  Various  groups  of  muscles  are 
affected  in  tetanic  seizures.  The  muscles  of  the  jaw,  if  affected, 
cause  trismus;  if  those  of  the  back  are  involved  the  individual  suffers 
from  opisthotonos.  The  seizures  may  be  constant  or  tonic;  or 
convulsive  and  violent,  then  they  are  designated  as  clonic. 

Immunity. — Metchnikoff  claims  that  the  only  natural  immunity 
possessed  by  man  against  tetanus  resides  in  his  leucocytic  powers 
of  defense.  Susceptibility  of  the  natural  receptors  of  the  nerve 
cells  for  the  toxin,  and  the  degree  of  affinity,  constitutes  the  cause 
of  intoxication,  its  degree,  and  ultimate  result.  Affinity  for  the 
receptors  of  other  less  vital  organs,  on  the  part  of  the  toxin,  estab- 
lishes a  means  of  natural  defense.  Acquired  immunity  is  dependent 
upon  the  formation  of  anti-toxin.  The  anti-toxin,  formed  by  suscep- 
tible animals  injected  with  tetanus  toxin,  is  chiefly  useful  and  valu- 
able as  a  prophylactic  measure.  An  epidemic  of  puerperal  tetanus 
in  a  lying-in  hospital  was  checked  by  its  use.  Sprinkling  dry  pow- 
dered anti-toxic  serum  on  wounds  infected  with  tetanus  bacilli,  or 
toxin,  prevented  infection  (Calmette  and  McFarland).  The  anti- 
toxin may  be  injected  either  into  the  substance  of  the  brain  in  cases 
of  well  developed  tetanus,  or  into' the  cerebro-spinal  fluid,  in  the 
hope  of  neutralizing  the  toxin  not  already  in  firm  combination  with 
the  nervous  elements.  Large  nerves  near  the  infecting  wound  may 
be  injected  with  anti-toxin  in  the  hope  of  binding  the  toxin  already 
in  combination  with  the  nerve  cells. 

Female  mice  immunized  against  tetanus  toxin,  transmit  a  great 
amount  of  immunity  to  their  off-spring.  The  milk  of  an  immunized 
mouse  also  causes  a  passive  immunity  in  other  young  that  are 
suckled  by  her. 


BACILLUS   OF   MALIGNANT   CEDEMA  1 73 

BACILLUS  OF  MALIGNANT  (EDEMA. 

Bacillus  CEdematis  Maligni. 

Vibrion  septiquh. 

Bacillus  of  Malignant  (Edema. 

Morphology  and  Stains. — Thickish  rods,  resembling  tetanus 
and  symptomatic  anthrax  baciUi,  with  a  tendency  to  grow  in  long 
threads.  It  is  actively  motile,  and  is  possessed  of  numerous  peri- 
trichous  flagella.  Spores  are  found  which  may  be  either  equatori- 
ally  or  polarly  situated.  This  organism  is  readily  stained  by  the 
ordinary  methods,  but  not  by  Gram's. 

Chemical  Activities, — Milk"  is  coagulated,  but  not  soured,  and 
the  reaction  is  amphoteric.  Abundant  alkaU  is  formed  at  times; 
albumin  is  decomposed,  forming  fatty  acids,  leucin,  an  oil,  and  an 
offensive  odor.     COjN.  and  marsh  gas,  are  also  formed. 

Habitat. — It  is  found  in  soil,  dust,  manure  and  dirty  water  and 
is  widely  distributed. 

Cultures. — This  organism  is  a  strict  anaerobic,  and  grows  well 
in  most  culture  media,  at  incubator  or  room  temperature.  On  gel- 
atine plates  colonies  develop  on  the  surface  (under  hydrogen) 
in  tiny  shining  white  bodies,  which  upon  magnification  are  found 
to  be  filled  with  a  grayish- white  substance  composed  of  melted  gela- 
tine, and  long  tangled  filaments.  The  edges  of  the  colonies  are 
fringed.  In  gelatine  stab  cultures  (made  in  liquid  gelatine,  which, 
after  inoculation,  is  rapidly  solidified  in  ice  water)  a  globular  area 
of  liquefaction  occurred.  If  sugar  is  added,  active  fermentation 
takes  place,  with  the  production  of  large  amounts  of  offensive  gas. 
It  grows  well  on  agar,  in  bouillon,  and  in  milk. 

Pathogenesis. — Is  pathogenic  for  man,  horses,  sheep,  dogs,  rab- 
bits, calves,  pigs,  goats,  rats,  mice,  and  guinea  pigs.  Cattle  are 
said  to  be  immune.  When  bacilH  are  appHed  to  a  scratched  sur- 
face, infection  is  not  likely  to  occur,  as  free  oxygen  seems  to  inhibit 
the  growth;  if,  however,  the  wound  is  deep,  rapid  infection  follows, 
young  domestic,  and  laboratory  animals  dying  within  forty-eight 


174  BACTERIA 

hours.  In  man,  the  clinical  manifestation  of  infection  with  this 
organism  is  known  as  maUgnant  cedema.  Infection  has  followed 
penetrating  wounds  of  the  body,  by  dirty  tools,  nails,  splinters, 
bullets,  etc.  The  disease  is  often  quickly  fatal.  It  produces,  fre- 
quently, rapid  moist  gangrene. 

Bacilli  have  been  found  in  the  blood  of  dead  animals.  Infection 
is  very  apt  to  follow  contused  wounds,  especially  if  other  bacteria, 
like  the  Bacterium  vulgaris,  or  Bad.  prodigiosus,  are  present.  A 
mixed  culture  in  vitro  of  this  organism,  and  the  Bacillus  acidi  para- 
lactici  produces  butyl  alcohol  abundantly.  Neither  of  these  organ- 
isms separately  can  do  so.  The  organisms  excrete  a  toxin  and 
animals  can  be  immunized  with  it.  One  attack  of  the  disease 
confers  immunity. 

SYMPTOMATIC  ANTHRAX  BACILLUS. 

Bacillus  Chauvoei. 

Bacillus  of  Symptomatic  Anthrax. 

Rauschhrand  Bacillus.     (Figs.  59  and  60.) 

The  cause  of  symptomatic  anthrax,  black-leg,  or  quarter-evil,  in 
cattle. 

Morphology  and  Stains. — This  is  a  large  organism,  .5/1  in  width, 
and  3  to  5//  in  length.  It  has  rounded  ends,  and  grows  in  pairs, 
but  not  in  strings  or  chains.  It  is  motile,  and  has  many  peritrichous 
flagella.  When  stained  for  spores,  these  bodies  may  be  found  dis- 
tending the  organism  in  the  middle  or  at  the  end,  and  the  bacillus 
assumes  a  drum-stick,  or  spindle  shape.  Often  chromophilic  gran- 
ules are  present;  involution  forms  also  appear,  and  are  of  enormous 
size.  This  organism  stains  with  all  the  common  stains,  but  not  by 
Gram's  method.  They  may  be  seen  in  an  unstained  condition  in 
blood  or  other  fluids. 

Habitat. — This  bacillus  is  found  not  only  in  the  diseased  tissues 
and  dead  bodies  of  infected  animals,  but  also  in  infected  pastures, 
soil,  hay,  etc. 


SYMPTOMATIC  ANTHRAX  BACILLUS  1 75 

Temperature  Requirements. — It  is  best  cultivated  at  body  tem- 
perature, but  grows  anywhere  between  i8°  C.  and  37°  C. 


Fig.  59. — Rauschbrand  bacilli  showing  spores.     (KoUe  and  Wassermann.) 


Fig.  60. — Rauschbrand  bacillus  showing  flagella.     (Kolle  and  Wassermann.) 

Cultures. — It  is,  like  tetanus  and  malignant  oedema  organisms, 
a  strict  anaerobe.  On  gelatine  it  grows  in  roundish  whitish  colonies 
in  a  delicate  tangled  mass,  with  projecting  filaments.     The  gelatine 


176  BACTERIA 

is  liquefied,  and  bubbles  of  gas  are  formed  in  stab  cultures.  A  sour 
odor  is  emitted  from  cultures;  i  percent  to  2  percent  of  sugar  is 
required  for  successful  cultivation;  or  5  percent  of  glycerine  will 
answer.  On  agar  the  growth  is  marked;  gas  is  produced,  and 
acidous  odors  evolved.  In  bouillon  it  grows  rapidly.  Large 
masses  of  the  organism  sink  to  the  bottom,  gas  is  formed,  and  the 
medium  is  clouded.  Milk  affords  a  good  medium  for  the  growth 
of  the  organism,  but  the  casein  is  not  coagulated. 

Pathogenesis. — Young  cattle,  six  months  to  four  years  old, 
sheep,  goats,  rats,  mice,  and  more  especially  guinea  pigs,  are  sus- 
ceptible to  it.  Swine  are  immune,  while  dogs,  cats,  birds,  and  rab- 
bits are  not  susceptible.  Man  is  immune.  It  causes  in  animals 
peculiar  groups  of  emphysematous  crepitating  pustules,  followed 
by  emaciation  and  death.  These  areas  contain  dark  fluid,  probably 
broken-down  blood.  In  guinea  pigs  inoculation  is  followed  by 
death  within  thirty-six  hours.  The  site  of  inoculation  is  found  to  be 
oedematous,  and  contains  bloody  fluid.  The  organs  generally 
are  normal.  The  bacilli  are  mostly  found  at  the  site  of  the  inocu- 
lation, but  later  in  the  blood  in  every  part  of  the  body.  The  viru- 
lence of  this  organism  in  culture  media  is  soon  lost.  The  addition 
of  lactic  acid  to  the  cultures  increases  their  virulence. 

Immunity. — It  is  possible  to  decrease  the  virulence  of  this  organ- 
ism, and  to  use  the  weakened  bacteria  as  a  vaccine  against  infection. 
To  attenuate  this  bacillus,  prolonged  exposure  to  heat,  or  to  heat 
and  drying  together  is  necessary.  Inoculation  with  bacilH  treated 
in  this  way  is  followed  by  a  mild  local  reaction,  which  affords  com- 
plete immunity  against  infection  with  virulent  bacilli.  It  has  been 
found  by  Kitt  that  the  muscles  of  an  infected  animal,  if  subjected 
to  a  high  temperature — 85°  C.  to  90°  C. — afforded  complete  protec- 
tion to  the  animal  inoculated  with  them.  It  is  best  to  use  a  weaker 
vaccine  muscle  that  has  been  heated  to  100°  C.  for  two  hours,  in 
order  to  protect  against  the  active  vaccine.  Before  heating,  the 
meat  is  ground.  When  used  as  an  injection,  it  is  crushed  and 
mixed  in  a  mortar  with  sterile  water.     Guillod  and  Simon  found 


MEAT  POISONING   BACILLUS  1 77 

that  this  means  of  preventative  inoculation  reduced  the  death  rate 
in  unprotected  animals  from  5-20  percent  to  5  percent.  If  this 
bacillus,  and  the  prodigiosus  bacillus  are  injected  into  naturally 
immune  animals,  death  will  often  result. 

There  is  a  soluble  toxin,  anti-toxin  against  which  appears  in 
immunized  animals.  The  toxin  may  be  used  for  prophylaxis.  One 
attack  confers  immunity. 

MEAT  POISONING  BACILLUS. 

Bacillus  Botulinus.     Van  Ermengen. 

Bacillus  of  Meat  Poisoning,  or  Botulism.     (Fig.  61.) 

Morphology  and  Stains. — This  bacillus  resembles  thick  vigorous 
rods,  4-9/jt  long,  and  9/1  thick,  is  motile,  has  polar  spores,  and 
from  four  to  nine  peritrichous  flagella.  It  is  strangely  called  a 
saprophyte,  because  it  is  incapable  of  growth  in  the  body,  yet  its 
toxin  is  highly  poisonous  to  man  and  other  animals.  It  is  stained 
by  all  the  usual  basic  aniline  dyes,  but  not  by  Gram's  method. 

Habitat. — Is  found  in  raw  meat,  improperly  cured  hams,  and  in 
sausages.     It  gains  access  to  meat  after  the  death  of  the  animal. 

Vital  Characteristics. — Is  an  anaerobe.  Its  thermal  death- 
point,  for  a  spore-bearing  organism,  is  low,  80°  C,  for  an  hour. 
Grows  only  in  media  that  are  alkaline,  and  is  capable  of  growth  at 
from  18°  C.  to  35°  C.,  though  best  below  35°  C;  6  percent  of 
chloride  of  soda  checks  growth. 

Chemical  Activities. — Can  produce  toxin  (which  is  soluble  in 
water)  at  a  relatively  low  temperature.  Milk  is  not  coagulated, 
grape  sugar  is  fermented,  and  a  foul,  sour  odor  is  produced  in  a 
culture.     It  liquefies  gelatine. 

Cultures. — On  gelatine  plate,  that  contains  sugar,  colonies  are 
produced  that  are  coarse  and  prickly  in  appearance.  The  lique- 
faction of  the  gelatine  is  slow.  Bouillon  is  rendered  turbid.  The 
cultures  resemble  tetanus  and  malignant  oedema. 

Pathogenesis. — Its  pathogenic  action  is  marked,  but  only  by  its 


1 78  BACTERIA 

toxin,  which  has  a  decided  affinity  for  nervous  tissue.  The  toxin 
is  absorbed  from  the  intestinal  tract  unchanged  by  the  gastric  juice. 
In  this  it  difiFers  from  the  toxin  of  diphtheria  and  tetanus.  If  the 
toxin  is  mixed  with  the  emulsified  nerve  tissue,  it  becomes  neutra- 
lized. In  fatal  cases  of  infection,  the  ganglionic  nerve  cells  are  de- 
generated. Man  is  very  susceptible,  while  cats  and  dogs  are  more 
or  less  non-susceptible.     If  bacilli  are  inoculated  into  animals,  they 


Fig.  6i. — Bacillus  of  botulism.     (KoUe  and  Wassermann.) 


do  not  proliferate.     Animals  that  recover  are  found  to  have  devel- 
oped strong  anti-toxin  in  the  blood  serum. 

Immunity. — ^An  artificially  prepared  anti-toxin  has  been  found 
to  be  active,  and  is  of  use  in  treating  cases  of  poisoning  with  meat. 

GASEOUS  EDEMA  BACILLUS. 

Bacillus  Capsulatus  Aerogenes. — Welch. 

Morphology  and  Stains. — A  vigorous  plump  bacillus  3  to  4/i  in 
length,  resembHng  the  anthrax  bacillus,  and  is  usually  straight. 
It  forms  spores,  is  non-motile,  and  flagella  have  not  been  found. 
It  occurs  in  pairs,  and  in  chains.     In  old  cultures  involution  forms 


GASEOUS  EDEMA  BACILLUS 


179 


are  seen.  Spores  are  generally  equatorially  situated.  Is  colored 
by  all  the  basic  dyes,  and  holds  the  stain  in  Gram's  method.  Stain- 
ing shows  that  it  possesses  a  capsule. 

Habitat. — The  soil,  the  intestines,  and,  sometimes,  the  skin  of 
man. 

Vital  Characteristics. — ^Vital  resistance  is  low,  the  thermal 
death-point  being  58°  C.  with  ten  minutes'  exposure.     It  grows 


r^-^ 

'^^n^ 


Fig.  62. — B.  Aerogenes  capsulatus  of  Welch,  in  smear.     (Williams.) 


best  at  body  temperature.  Has  Hved  for  one  hundred  days  on 
culture  media  in  the  incubator.     It  is  an  anaerobe. 

Chemical  Activities. — ^Produces  gas;  does  not  usually  hquefy 
gelatine,  but  curdles  milk.     (Fig.  63.) 

Cultures. — Grows  best  in  neutral  or  alkaUne  media,  producing 
abundant  gas.  Colonies  appear  grayish  or  brownish- white,  and  are 
often  surrounded  by  projections  which  are  feathery  or  hair-like.     On 


i8o 


BACTERIA 


agar  strict  anaerobic  conditions 
are  necessary  for  growth,  gas  bub- 
bles appear  in  the  media,  and 
the  agar  may  be  forced  out  of  the 
tube  in  stab  cultures.  In  bouillon 
it  grows  under  anaerobic  condi- 
tions. The  growth  is  rapid, 
bouillon  is  clouded,  and  a  froth 
appears  on  the  surface.  After  a 
few  days  the  media  becomes  clear, 
owing  to  the  sedimentation  of  the 
bacilli.  Growth  occurs  best  in 
sugar  bouillon,  which  becomes 
strongly  acid.  In  milk  the  growth 
is  rapid  and  luxuriant;  the  pro- 
teids  are  coagulated.  Anaerobic 
conditions  must  be  observed.  On 
potato  it  grows  well,  producing 
bubbles  in  the  water  which  may 
cover  the  potato  in  the  tube.  The 
growth  appears  thin,  moist,  and 
grayish-white. 

Pathogenesis. — The  pathogenic 
properties  of  this  organism  are 
limited.  It  is  not  able  to  endure 
the  oxygen  of  the  circulating  blood. 
Grows  best  in  old  clots,  and  in 
the  uterus.  It  produces  gas  rapidly 
in  some  cases  of  abortion  and  in 
peritonitis  in  man,  which  is  quickly 
followed  by  death.  It  causes  gase- 
ous phlegmons  in  guinea  pigs,  and 

injections  are    usually    fatal  to 

Fig.  63. — B.  Aerogenes  capsulatis,  ,  .    ,         ,                  •    r     x-         x.  r  ^ 

agar  culture  showing  gas  formation,  birds.     In  man  mfection   has  fol- 
(Williams.) 


CHOLERA    BACILLUS  l8l 

lowed  wounds,  and  delivery  of  the  child  in  puerperal  cases. 
It  produces  in  fatal  cases  the  condition  known  as  frothy  organs 
— "Schaumorgane."  It  may  be  isolated  from  infected  mat- 
ter, feces  etc.,  by  injecting  the  latter  into  a  rabbit's  vein  and  then 
killing  the  animal.  The  carcass  is  then  placed  in  an  incubator 
and  an  enormous  growth  of  the  organisms  follows;  anaerobic  condi- 
tions favorable  to  growth  are  obtained  in  the  blood;  from  the  latter 
pure  cultures  are  easily  obtained. 

Another  spore  forming  anaerobe  very  close  to  Welch's  bacillus 
is  called  Bacillus  enteritidis  sporogenes.  Its  differentiation  is 
probably  certain  but  difficult  to  make. 

Vincents  Angina  is  due  to  an  anaerobic  organism  of  two  stages, 
as  Bacillus  fusiformis  and  Spirochceta  vincenti.  The  bacillus  is  a 
fusiform  irregularly  staining  pointed  rod,  3-1 2/x  long  by  .3-8// 
wide.  Under  cultivation  it  grows  out  into  forms  such  as  are  seen 
with  it  in  smears  from  the  diseased  throat,  that  is,  long,  wavy,  uni- 
formly stained,  flexible,  pointed  ended  spirals.  The  bacillus  forms 
endospores  chiefly  at  the  end.  ObHgate  anaerobe,  requiring 
serum  ascitic  fluid  or  glycerin.  Colonies  delicate  and  whitish. 
Gas  in  glucose  media.  Litmus  milk  only  decolorized.  Gives  a 
fetid  odor  on  all  cultures.     No  specific  immunity  reactions  known. 

SPIRILLACEiE. 

CHOLERA  BACILLUS. 

Vibrio  Cholerae.     Koch. 

Spirillum  CholercB.   ,  (Fig.  64.) 

Cholera  Bacillus. 

Comma  Bacilhis. 

Morphology  and  Stains. — Curved  or  bent  rods,  the  ends  not 
lying  in  the  same  plane.  This  bending  varies  greatly.  Under 
certain  conditions  of  growth  such  as  the  presence  of  alcohol,  or  in- 
sufficient albumin  or  oxygen  in  culture  media,  long  spiral  chains 
are  formed.     It  is  motile,  has  one  terminal  flagellum,   and  like 


I 82  BACTERIA 

Other  members  of  this  family,  has  no  spores.  It  stains  well  with 
the  common  dyes  but  not  by  Gram's  method.  Dilute  fuchsin 
stains  it  best.  Occasionally  involution  forms  are  developed,  which 
do  not  stain  well.  So-called  arthrospores  are  formed,  according  to 
Hiippe. 

Habitat. — It  is  said  to  exist  constantly  in  the  waters  of  the  Gan- 
ges in  India.  Is  frequently  found  in  contaminated  drinking  water, 
from  rivers,  lakes,  and  wells;  also  in  human  feces,  which,  used  as 


Fig.  64. — Cholera  spirilla.     (Kolle  and  Wassermann.) 

manure,  infests  vegetables,  and  spreads  the  disease.  It  is  found 
in  the  intestines  during  cholera,  and  after  death  in  other  viscera. 

Vital  Resistance. — Is  extremely  sensitive  to  various  deleterious 
agencies.  Minute  quantities  of  mineral  acids,  and  other  chemical 
disinfectants,  as  well  as  light,  heat,  and  drying,  quickly  kill  it; 
one  percent  carbolic  kills  rapidly.  A  1-2,000,000  solution  of  cor- 
rosive subHmate  destroys  in  from  five  to  ten  minutes.  Its  thermal 
death-point  is  60°  C.  for  ten  minutes  (moist  heat). 

Chemical  Activities. — It  creates  indol  in  large  quantities,  and 
may  be  detected  in  peptone  cultures  merely  by  the  addition  of  sul- 


CHOLERA  BACILLUS  1 83 

phuric  acid.  Dextrorotatory  lactic  acid  is  produced  from  all  the 
sugars.  Gases  are  not  formed.  Yields  alkali  in  culture;  causes 
slight  coloration  of  potato,  and  produces  a  disagreeable  odor  in 
bouillon;  also  yields  HjS,  and  ferments  that  liquefy  gelatine. 
Bacteriolysins  and  invertin  are  also  produced,  as  well  as  a  toxin 
which  is  soluble  in  water.  The  most  powerful  toxin,  by  far,  is 
contained  in  the  cells  of  the  vibrio  themselves.  This  causes  death 
after  intra-peritoneal  injection  in  guinea  pigs. 

Oxygen  Requirements. — It  is  a  facultative  aerobe;  its  growth, 
however,  without  oxygen  is  slow,  while  powerful  toxins  are  formed. 

Temperature. — Grows  best  at  37°  C,  but  very  well  at  23°  C. 
Does  not  grow  below  8°  C. 

Cultures. — On  gelatine  plates  the  growth  is  characteristic. 
Small  yellowish-white  colonies,  which  rapidly  liquefy  the  gelatine, 
appear  in  twenty-four  hours.  As  the  colony  increases  in  size  it 
becomes  more  and  more  granular,  and  finally  the  whole  medium  is 
liquefied.  In  gelatine  tube  stab  culture,  the  growth,  at  first,  is 
not  characteristic;  but,  after  a  few  hours,  a  semi-spherical  depres- 
sion appears,  which  extends  downward,  and  resembles  a  large 
bubble  of  gas.  As  liquefaction  progresses,  the  whole  line  of  punc- 
ture disappears,  and  the  excavation  looks  cylindrical.  This  area 
becomes  cloudy.  On  agar  plates  the  colonies  are  elevated,  round 
and  white,  with  a  moist  lustre.  Deep  colonies  are  whetstone  shape. 
Old  agar  colonies  become  yellowish-brown.  Coagulated  blood 
serum  is  rapidly  liquefied  at  37°  C.  Milk,  at  times,  is  coagulated. 
No  curdling  ferment  is  formed;  the  acid  produced  is  thought  to  be 
sufficient.  On  potato  the  growth  is  slow,  or  not  at  all,  if  the  medium 
is  acid.  If  the  potato  is  rendered  alkahne,  growth  occurs,  with  a 
moist  lustre,  slightly  elevated;  white  at  first,  later  becoming  brown. 
On  acid  fruits  it  will  not  grow.  In  bouillon,  after  sixteen  hours, 
a  diffuse  cloudiness  occurs,  with  the  formation  of  a  stiff  pelHcle, 
which  in  some  cultures  becomes  wrinkled.  In  peptone,  abundant 
growth  takes  place,  with  the  production  of  indol  and  nitrites.  If  a 
few  drops  of  H3SO4  are  added,  a  beautiful  red  appears  if  nitrites  are 


184  BACTERIA 

present.  This  is  the  "cholera  red  ".reaction.  If  the  color  does  not 
at  once  appear,  nitrites  must  be  added. 

Pathogenesis. — Cholera  spirilla  are  pathogenic  for  man  and 
guinea  pigs.  If  the  stomach  of  the  latter  is  rendered  alkaline  with 
bicarbonate  of  soda,  and  a  bouillon  culture  introduced,  choleraic 
symptoms  will  follow  and  the  animal  will  die.  If  cholera  spirilla 
are  injected  into  the  peritoneum,  the  animal  will  quickly  succumb 
to  a  general  cholera  peritonitis.  Young  rabbits  are  equally  suscep- 
tible. When  cholera  spirilla  in  culture  have  been  swallowed  by 
man  (laboratory  workers) ,  either  by  design  or  accident,  the  disease 
has  followed,  sometimes  with  fatal  results.  The  toxin  of  this  organ- 
ism is  intra-cellular  (an  endo-toxin).  Old  cultures  become  patho- 
genic through  a  bacteriolytic  action,  by  which  the  cells  are  dis- 
solved, and  the  toxin  liberated.  Filtrates  from  young  cultures  are 
non-toxic.  If  bouillon  cultures  are  killed  by  chloroform,  and  then 
injected  into  animals,  toxic  action  follows.  In  cholera  the  patho- 
genic process  is  mostly  confined  to  the  intestines.  Toxic  absorp- 
tions, due  to  the  liberation  of  toxic  products  by  the  bacteriolytic 
action  of  serum,  follow  later.  There  is  a  desquamation  of  the 
epithelium  of  the  bowel,  and  epithelial  cells  found  in  the  watery 
discharges  resemble  rice  grains.  Peyer's  patches  may  become 
slightly  swollen  and  reddened,  and  later,  there  may  be  a  diphtheritic 
necrosis  above  the  iliocecal  valve,  and  often  a  parenchymatous 
nephritis.     The  vibrios  do  not  enter  the  blood. 

Diagnosis. — Bacteriological  diagnosis  of  cholera  is  accomplished 
by  examining  the  alvine  discharges.  A  mucous  flake  is  mixed  with 
some  peptone  solution,  this  is  incubated,  and  the  spirilla,  if  present, 
rapidly  grow  on  the  surface;  after  a  few  hours,  plates  are  poured 
from  this  surface  growth,  and  from  the  plates  liquefying  colonies 
are  picked  out,  and  bouillon  cultures  made.  These  are  tested 
by  dried  serum,  from  horses  artificially  immunized  by  injecting 
cholera  spirilla  into  them.  If  the  organism  under  examination 
(after  serum  mixed  with  2,000  to  3,000  parts  of  water  is  added) 
agglutinates,  it  is  considered  to  be  the  cholera  spirillum.     Both  in 


GLANDERS   BACILLUS  1 85 

early  and  fatal  cases,  the  agglutinating  reaction  is  not  available, 
since  it  takes  some  time  for  the  agglutinins  to  form  in  the  blood. 
Under  the  chapter  on  immunity  an  account  of  the  Pfeiffer  reaction 
is  given,  also  one  on  vaccination  against  cholera  infection,  by  means 
of  killed  cultures,  under  the  chapter  on  vaccines. 

Vibrios  Allied  to  the  Cholera  Vibrio. 

Several  other  vibrios  have  been  discovered  that  resemble  the 
cholera  vibrio.  These  are  mostly  found  in  potable  waters,  and 
though  in  many  respects  identical  with  the  cholera  vibrio,  they 
differ  in  essential  points,  i.e.,  pathogenicity,  and  in  their  agglutina- 
bility  with  specific  sera.  The  most  important  of  these  organisms 
are:  Vibrio  Metchnikovii;  Vibrio  proteus;  Vibrio  tyrogenum;  and 
Vibrio  sclmylkilliensis.  There  are  no  important  pathogenic  mem- 
bers of  this  group  except  the  cholera  vibrio. 

GLANDERS  BACILLUS. 

Bacterium  Mallei. 

Bacillus  Mallei. 

Glanders  Bacillus. 

Morphology  and  Stains. — Slender  rods  2  to  3//  in  length,  con- 
taining no  true  spores,  but  shining  chromatophilic  bodies  {Babes- 
Ernst  granules).  In  old  culture,  long  club-like  threads  appear, 
which  exhibit  true  branching.  This  organism  is  not  motile,  and 
has  no  flagella.  It  is  stained  with  difficulty  by  ordinary  methods, 
and  not  at  all  by  Gram's  method. 

Vital  Activities. — It  is  a  facultative  aerobe,  growing  feebly  in 
the  absence  of  air,  and  best  at  37°  C,  in  glycerine  agar.  Resists 
drying  but  feebly.  Its  thermal  death-point  is  55°  C,  10  minutes' 
exposure. 

Chemical  Activities. — ^Produces  a  brown  pigment  on  potato,  also 
mallein,  and  a  httle  indol  in  old  bouillon  cultures.     It  forms  no  gas. 


I 86  BACTERIA 

Cultures. — On  gelatine  it  produces  small  punctiform  colonies 
that  are  white,  and  become,  after  a  time,  surrounded  by  a  distinct 
halo.  The  colonies  are  often  very  deHcate  and  ragged.  The  gel- 
atine is  not  liquefied.  On  agar  the  growth  is  best  if  glycerine  is 
present,  but  is  not  characteristic.  Bouillon  cultures  cause  an 
abundant  sediment,  above  which  the  medium  is  clear.  Milk  is  co- 
agulated. On  potato  the  growth  is  characteristic.  The  color  is, 
at  first,  yellowish-white  like  honey,  becoming,  finally,  reddish- 
brown.     The  potato  is  much  darkened. 

Pathogenesis. — This  organism  is  pathogenic  for  horses  and  man; 
50  percent  of  men  succumb  after  infection.  Horses,  asses,  cats, 
dogs,  sheep,  and  goats  are  susceptible  in  the  order  mentioned. 
Cattle  and  birds  are  immune.  In  horses  the  disease  is  known  as 
glanders,  or  farcy,  and  the  avenue  of  infection  determines  the  clin- 
ical form  of  the  disease.  The  mucous  membrane  and  the  skin  are 
the  chief  places  of  infection.  A  primary  ulcer  is  formed  in  the 
mucous  membrane  of  the  nose,  or  in  the  skin.  Subsequently,  the 
lymph  glands  and  the  lungs  may  be  infected.  Guinea  pigs  are 
easily  infected.  White  and  gray  mice,  and  rats  are  immune.  For 
purposes  of  diagnosis  guinea  pigs  are  inoculated,  but  care  must  be 
used,  as  several  fatal  cases  have  occurred  in  laboratory  workers, 
it  being  a  treacherous  organism  with  which  to  work.  In  infected 
animals,  it  produces  a  rapid  and  marked  inflammatory  reaction, 
with  the  formation  of  pus.  Certain  "buds,"  or  nodules  are  formed, 
which  are  between  an  abscess  and  a  tubercle  in  structure. 

The  diagnosis  of  doubtftil  cases  may  be  made  by  injecting  the 
material  into  the  peritoneum  of  male  guinea  pigs.  A  violent 
suppurative  orchitis  occurs  from  which  the  rods  can  be  cultivated. 
The  poisons  are  endotoxic^. 

Agglutinations. — It  has  been  shown  by  McFadyean  that  the 
blood  of  infected  horses  exhibits  markedly  agglutinative  properties 
toward  the  glanders  bacilli.  A  slight  immunity  is  present  after 
an  attack. 

Mallein. — In  old  cultures  a  peculiar  tuberculin-like  substance 


DIPHTHERIA  BACILLUS  1 87 

(mallein)  is  formed  from  the  bodies  of  the  bacilli  themselves,  and 
in  the  bouillon.  This  is  thermostabile  and  if  injected  into  animals 
having  glanders,  produces  a  marked  reaction.  Locally,  if  the  horse 
is  glanderous,  a  hard  swelHng  is  formed,  and  the  temperature  is 
raised  1.5°  C.  to  2°  C.  in  a  few  hours.  This  is  considered  a  valuable 
means  of  diagnosis  by  veterinarians. 

DIPHTHERIA  BACILLUS. 

Corynebacterium  Diphtherise.     (Loffler.) 
Bacillus  Diphthericd. 
Klehs-Loffler  Bacillus. 
Diphtheria  Bacillus. 

Morphology  and  Stains. — Long,  bent,  or  curved  bacilli  of  irreg- 
ular contour,  frequently  clubbed  or  filiform  at  one  or  both  ends; 


Fig.  65. — Diphtheria  bacilU  in  mucus  of  trachea  stained  with  fuchsin.     (Kolle 
and  Wassermann.) 

which  contain  chromatophilic  granules,  and  often  exhibit  true 
branching;  have  no  spores  or  flagella,  and  are  not  motile.  Accord- 
ing to  Wesbrook,  stained  bacilli  are  of  three  types:  (i)  granular 
(containing  the  Babes-Ernst  granules);  (2)  barred  like  a  striped 


I 88  BACTERIA 

stocking;  or  (3)  solid,  staining  uniformly  throughout.  The  pleo- 
morphic differences  of  various  bacilli  are  most  characteristic,'  and 
of  diagnostic  importance.  This  organism  stains  with  all  the  basic 
dyes,  notably  by  Loffler's  blue,  or  Neisser's  special  granule  stain. 
It  is  also  stained  by  Gram's  method.  The  length  of  the  organism 
differs  much,  according  to  the  reaction  of  the  medium  in  which  it 
grows.  Alkaline  media  favor  long  forms,  and  acid  the  reverse. 
Its  length  is  from  1.5/z  to  3.5/^.  It  does  not  form  chains.  Bizarre, 
or  involution  shapes  predominate  in  old  cultures.     (Fig.  66.) 


Fig.  66. — Diphtheria  bacilli  involution  forms.     (Kolle  and  Wassermann.) 

Culture  and  Temperature  Requirements. — It  grows  best  at 
body  temperature,  and  on  glycerine  agar,  or  in  LoflBler's  blood 
serum  mixture  of  alkaline  reaction. 

Vital  Characteristics. — It  resists  drying  for  a  long  time,  and  has 
lived  on  culture  media  for  eighteen  months  at  room  temperature; 
also  in  silk  threads  for  several  months  in  a  dried  condition.  Re- 
mains alive  in  healthy  throats  for  months.  Formalin  vapor  kills 
it  speedily;  corrosive  subUmate  solution,  1-10,000  destroys  it  in  a 
few  minutes;  light  is  lethal  to  it  in  from  two  to  ten  hours,  and  heat 
at  58°  C.  in  ten  minutes. 


DIPHTHERIA   BACILLUS  1 89 

Habitat. — It  has  not  been  found  in  sewage,  or  sewer  gas,  soil 
or  water,  the  disease  therefore  is  never  transmitted  by  these  means. 
Has  been  found  in  the  throat,  nose,  and  in  the  conjunctivae  of 
healthy  bodies.  In  disease,  the  organism  is  mostly  found  in  the 
throat,  but  has  been  isolated  from  all  the  organs  in  some  fatal 
cases.  Sometimes  it  is  discovered  in  the  throats  of  animals. 
Though  its  action  is  local,  it  elaborates  a  toxin  which  acts 
systemically. 

Cultures. — On  gelatine  plate  the  growth  is  scanty  and  raised. 
This  medium  is  never  used  for  cultivating  this  organism.  The  gela- 
tine is  not  liquefied.  On  glycerine  agar  plates  the  growth,  though 
moderate,  is  typically  characteristic,  but  very  slightly  raised  above 
the  medium,  and  is  of  duller  lustre.  Old  colonies  become  yellowish- 
brown,  the  center  of  which,  under  a  magnification  of  sixty  diameters, 
appears  darker,  and  with  ravelled  edges.  On  Loffler's  blood 
serum  mixture,  the  organism  grows  rapidly  and  well.  This  and 
ascites-glycerine-agar  culture  media  are  the  best  for  it.  Bouil- 
lon made  from  fresh  meat  is  an  excellent  medium  for  its  growth. 
The  bouillon,  which  must  be  alkaline  and  freshly  made,  becomes 
first  cloudy;  then  a  fine  precipitate  settles,  and  over  the  surface  a 
delicate  pellicle  forms.  The  reaction  of  the  culture  presents  three 
types:  A,  is  acid  in  the  beginning,  and  becomes  progressively  more 
acid.  B,  is  alkaline  from  the  start,  and  progressively  more  alkaline; 
this  is  the  most  toxic  growth.  C,  acid  at  the  start,  becoming 
alkaline  finally.  The  growth  is  not  so  luxuriant  as  in  B,  nor  is 
there  as  much  toxin  produced.  In  milk,  the  growth  is  luxuriant, 
without  coagulation.  The  reaction  is  amphoteric,  but  in  old  cul- 
tures it  becomes  alkaline.  On  potato,  rendered  alkaline,  it  will 
grow,  but  not  characteristically. 

Chemical  Activities. — No  gas  is  formed,  or  any  curdling  or 
gelatine  dissolving  ferments,  but  H2S,  and  indol,  are  produced. 
Acids  are  evolved  from  sugars;  even  the  sugar  found  in  meat  is 
converted  into  lactic  acid.  In  the  manufacture  of  toxin,  this  muscle 
sugar  must  -be  removed.     A  soluble  toxalbumin  is  created,  both 


I 90  BACTERIA 

in  the  body  and  in  culture,  which  is  intensely  poisonous.  See 
chapter  on  bacterial  products. 

Pathogenesis. — Diphtheria  in  man  means  generally  an  infection 
of  the  mucous  membrane  of  the  upper  respiratory  tract,  with  the 
formation  of  false  membranes.  The  latter  may  cause  death  by 
suffocation.  Infection  may  occur  in  the  skin,  vulva,  or  prepuce. 
The  toxin  not  only  causes  a  local  necrosis,  with  the  formation  of  an 
exudate,  consisting  of  fibrin  and  leucocytes,  but  also  grave  systemic 
action,  with  marked  degeneration  of  important  nerves  and  nerve 
centers,  and  also  of  the  parenchyma  of  the  kidneys,  liver,  and  heart, 
paralysis  following.  In  certain  structures  fragmentation  of  the 
nuclei  of  the  cells  is  noted.  Guinea  pigs,  cats,  horses,  and  cows, 
may  be  infected  artificially,  but  the  disease  never  occurs  spon- 
taneously in  these  animals.  Horses,  dogs,  and  cattle  are  susceptible 
to  its  toxin.  Diphtheria  bacilli  often  have  associated  with  them 
streptococci,  which  add  to  their  virulence,  and  complicate  the 
disease.  Endocarditis,  adenitis,  pneumonia,  abscesses,  and  empy- 
emia,  may  be  caused  by  them.  There  may  be  puerperal  diphtheria, 
due  to  the  infection  of  the  puerperal  tract.  Diphtheria  is  spread 
mostly  by  personal  contact  with  individuals  suffering  from  the 
disease,  or  with  convalescents,  in  whose  throats  virulent  bacilli 
linger,  perhaps,  for  months.  It  may  originate  from  infected  milk, 
contaminated  from  human  sources. 

Perhaps  the  most  important  source  of  infection,  especially  during 
an  epidemic,  is  the  healthy  bacillus  carrier  who,  wholly  unaware 
of  his  condition,  is  carrying  virulent  germs  in  his  throat.  This 
further  indicates  that  individual  resistance  or  susceptibility  plays 
an  important  part  in  infection. 

Immunity  is  natural,  active,  artificial,  or  passive.  Active  im- 
munity, following  infection,  is  generally  a  permanency,  for,  once 
infected,  the  individual,  if  he  recovers,  may  be  considered  immune 
for  a  time,  though  some  individuals  are  more  susceptible,  and  suffer 
several  attacks.  In  active  immunity  anti-toxin  is  found  in  the  blood, 
and  recovery,  and  subsequently,  immunity  is  due  to  this  fact.     Anti- 


DIPHTHERIA  BACILLUS  IQI 

toxin  may  be  discovered  in  the  blood,  by  mixing  it  with  toxin  of 
known  strength,  and  injecting  it  into  guinea  pigs.  If  these  survive 
a  large  lethal  dose  of  the  toxin,  it  is  safely  presumed  that  anti-toxin 
was  present  in  the  serum  abstracted. 

Passive  artificial  immunity  is  induced  by  injecting  anti-toxin  in 
the  bodies  of  persons  exposed  to  diphtheria.  It  is  most  effective 
but  is  short  lived,  lasting  only  a  few  weeks.  Serum  therapy  (see 
anti-toxin  in  previous  chapter).  If  there  is  one  natural  specific 
cure  for  any  disease,  it  is  diphtheritic  anti-toxic  serum,  which  is 
prepared  by  immunizing  horses  with  toxin,  and  abstracting  their 
blood.  This  is  measured  in  units,  i,ooo  to  5,000  units  forming 
a  dose.  The  earlier  it  is  given,  the  better  are  the  chances  of  recov- 
ery. As  a  prophylactic,  from  600  to  1,000  units  should  be  used. 
As  many  as  100,000  units  have  been  injected  in  a  single  patient.  No 
case  is  too  trivial,  or  too  far  advanced  in  which  to  use  it.  The 
serum  is  anti-toxic,  and  not  bactericidal. 
Wassermann  has  prepared  a  serum  that  is 
bactericidal,  and  is  designed  to  destroy  the 
bacilli. 

Pseudo-diphtheria  bacilli,  which  mor- 
phologically and  culturally  resemble  the  true 
bacilli,  have   been   described.     They  are  not 

pathogenic,  in  the  sense  of  producing  exuda-      Fig.  67.— Diphtheria 

J.    ,  ^,      .  J  1    V        J    i.      r         ^^         bacilli    stained    with 

tive  diphthena,  and  are  beheved  to  be  atten-  Loffler's  blue.  Striped. 

uated  diphtheria  baciUi  by  many  observers.  (Greene's  Medical 
The  diagnosis  of  diphtheria  by  culture  is  an  ^^S^osis. 
important  measure.  It  depends  upon  the  rapid  growth  of  the 
bacilli  upon  Loffler's  blood  serum.  Of  all  the  various  organisms 
found  in  the  throats  of  patients  with  diphtheria,  the  diphtheria 
bacilli  outstrip  them  in  rapidity  of  growth.  After  eight  to  twelve 
hours,  the  serum  inoculated  with  the  smear  from  the  false  mem- 
brane is  covered  with  fine  granular  colonies  of  pure  diphtheria 
bacilli.  After  twenty-four,  or  more  hours,  the  other  organisms 
present  overgrow  the  diphtheria  colonies.     A  sterile  swab  of  cot- 


192  BACTERIA 

ton,  or  a  stick,  is  rubbed  over  the  false  membrane,  or  throat,  and 
then  over  the  serum;  the  latter  is  incubated,  and  the  culture  ex- 
amined after  eight  or  twelve  hours,  by  staining  with  L6fl9er's  blue. 
If  curved,  clubbed,  irregularly  stained  bacilli  are  found,  especially 
if  they  contain  dark  polar  granules,  and  are  generally  uneven  in 
size  and  bizarre,  it  may  be  safely  considered  that  they  are  diphtheria 
bacilli.  Gram's  stain  may  be  needed  to  confirm  the  diagnosis 
occasionally,  or  it  may  be  necessary  to  inoculate  guinea  pigs. 

PSEUDO-DIPHTHERIA  BACILLUS. 

CorynebacteriumPseudo-diphtheriticum. 

Pseudo-diphtheria  Bacillus.     (Hoffmann.) 

Morphology  and  Stains. — This  bacillus  resembles  the  diph- 
theria bacillus.  The  rods,  however,  are  shorter  and  thicker;  other- 
wise, it  stains  like  the  true  bacillus,  but  not  by  Neisser's  method. 

Culture. — On  glycerine  agar  the  growth  becomes  diffuse, 
spreading  from  the  line  of  inoculation  in  a  grayish-yellowish  pasty 
expanse.  It  grows  well  on  gelatine.  In  bouillon  it  forms  a 
denser  and  more  luxuriant  growth  than  the  true  bacillus. 

Habitat. — It  is  found  in  healthy  throats  and  conjunctivae. 

Pathogenesis. — It  is  non-pathogenic  for  guinea  pigs.  It  can 
produce  abscesses,  nasal  sinusitis  and  otitis  media,  and  even  endo- 
carditis in  man. 

Diagnosis. — It  can  be  differentiated  from  the  true  bacillus  by 

1.  Being  non-pathogenic. 

2.  Not  exhibiting  polar  granules  with  Neisser's  stain. 

3.  Not  producing  acids  in  certain  carbohydrate  media. 
Bacillus  xerosis  is  a  pseudo-diphtheria  organism   found  on  the 

normal  conjunctiva.     It  is  not  thought  to  possess  any  virulence. 

TUBERCLE  BACILLUS. 

Mycobacterium  Tuberculosis. 

Bacillus  tuberculosis.     (Fig.  68.) 
Tubercle  bacillus. 


TUBERCLE   BACILLUS  1 93 

Morphology  and  Stains. — Slender  rods,  generally  unbranched, 
I.  .5/^  long,  and  .4/1  thick,  usually  slightly  bent;  are  non-motile,  and 
have  no  spores  or  flagella.  In  old  cultures,  and  sometimes  in 
sputum,  branching  forms  are  seen,  and,  rarely,  some  that  are  club- 
shape.  On  acid  potato,  thread  forms  are  found.  In  the  continuity 
of  most  of  the  bacilli,  unstained  spaces  are  seen;  in  others  dense 
deep  red  granules  are  found  by  fuchsin.  As  this  bacillus  is  difficult 
to  stain,  special  methods  have  been  devised  to  demonstrate  it,  as 
the  sheathing  capsule  renders  it  extremely  unsusceptible  to  the 
ordinary  methods  of  staining.  The  cause  of 
this  resistance  is  supposed  to  be  a  fatty  or 
v^axy  substance  in  the  capsule  which  is  more 
than  probable,  because  of  the  fact  that  stains 
that  are  fat  selective,  such  as  Sudan  III,  color 
it  very  v^rell.  Boiling  hot  carbol-fuchsin  gives 
it  the  best  stain.  It  keeps  the  color  in  spite 
of  the  action  of  strong  solutions  of  mineral  acids  Fig.  68. — Tubercle 
in  water,  or  dilute  alcohol.  So  when  tissues,  gained  ^with  ^uchsiii 
or  secretions,  are  stained  with  hot  carbol-  and  methylene  blue, 
fuchsin  for  a  short  time,  or  cold  carbol-fuchsin  i)iagnosis.) 
for  a  long  time,  and  then  treated  with  a  25 
percent  solution  of  HNO3,  or  H2SO4,  in  water,  everything  is 
deprived  of  the  red  color,  except  the  tubercle  bacilli.  All  such 
organisms  that  are  acid  proof,  are  called  "acid-fast."  There 
are  many  other  bacilli  that  have  this  property.  Aniline  water 
and  gentian  violet  solution  also  stain  it.  Gram's  method  dyes 
the  organism  violet.  Sometimes  very  young  bacilli  do  not  stain 
at  all. 

Vital  Requirements. — This  bacillus  thrives  best  at  37.5°  C.  It 
grows  slowly,  is  a  strict  parasite,  and  an  obligate  aerobe.  In  cul- 
tures it  dies  quickly  in  sunlight,  and  in  diffuse  daylight  it  dies  in  a 
few  days.  It  resists  drying  and  light  in  sputum  for  months.  Its 
thermal  death-point  (moist)  is  80°  C.  for  ten  minutes;  can  resist 
60°  C.  for  one  hour,  but  succumbs  to  95°  C.  in  one  minute.  It  is 
13 


194  BACTERIA 

quickly  killed  by  formaline  and  corrosive  sublimate,  but  resists 
3  percent  solution  of  carbolic  acid  for  hours.  In  sputum  it  with- 
stands antiseptics  for  a  long  time. 

Chemical  Activities. — It  grows  slowly,  producing  no  coloring 
matter;  yields  an  aromatic  sweetish  odor,  but  no  gas  or  acid.  It 
produces  certain  plasmins  or  endo-toxins,  which  are  called  tuber- 
culins (q.v.). 

Chemically  the  tubercle  bacillus  contains  two  fatty  matters,  one 
combined  with  an  alcohol  to  form  a  wax.  It  has  also  a  protamin, 
a  nucleic  acid  or  an  albumose.  Various  fatty  acids  are  to  be 
derived  from  it  by  chemical  treatment.  The  active  principle  in 
tuberculin  centers  around  its  protein  elements,  but  is  not  exactly 
known. 

Habitat. — It  is  a  strict  parasite  and  never  leads  a  saprophytic 
existence.  Is  found  wherever  human  beings  live  in  crowded 
quarters;  in  dust  of  rooms,  vehicles,  and  streets;  and  often  in  milk 
and  butter.  Has  also  been  observed  in  the  tissues  and  secretions 
of  non- tuberculous  persons.  It  is  very  widely  distributed,  being 
found  in  all  human  communities. 

Cultures. — Since  the  organism  does  not  grow  below  30°  C,  gela- 
tine is  never  used.  On  coagulated  blood  serum  of  cows,  horses, 
and  dogs,  this  bacillus  grows  best.  As  it  is  very  difficult  to  isolate 
in  pure  cultures,  the  following  procedure  should  be  followed:  The 
suspected  sputum,  fluid,  or  tissue  is  injected  into  a  guinea  pig,  and 
when,  in  two  weeks  or  more,  large  swollen  glands  can  be  felt  in  the 
groin,  the  ammal  should  be  killed,  and  a  gland  removed  under 
strict  aseptic  precautions.  It  is  then  divided,  and  the  halves  con- 
taining the  bacilli  are  rubbed  over  the  surface  of  coagulated  dog 
serum  and  allowed  to  remain  in  contact  with  it.  The  serum  should 
be  coagulated  in  special  tubes,  with  glass  caps,  having  small  per- 
forations, which  are  stopped  with  asbestos  fiber,  or  glass  wool.  The 
organism  grows  well  in  air,  but  too  great  access  thereto  dries  and 
kills  it.  After  the  tubes  are  incubated  for  a  week  or  two,  little 
scales  growing  into  clumps  appear,  which  are  lobulat^d  and  friable. 


TUBERCLE   BACILLUS 


195 


At  first  white,  it  later  turns  darker. 
This  medium  is  never  liquefied  by  the 
culture.  On  glycerine  agar  made  of 
veal  broth  containing  6  percent  of 
glycerine,  the  organism  grows  well 
after  isolation  from  the  tissues,  often 
luxuriantly.     (Fig.  69.) 

A  wrinkled  film  covers  the  surface 
of  the  agar,  from  which  it  is  removed 
with  ease.  On  bouillon,  made  of 
veal  and  glycerinized,  it  develops 
rapidly,  covering  the  medium  with  a 
dense  white  wrinkled  pellicle,  which, 
though  thick,  is  friable.  After  a 
time  it  falls  to  the  bottom  of  the  flask. 
It  grows  well  on  glycerinized  potato 
also,  and  milk  agar.  On  egg  albu- 
mins mixed  together,  sterilized  and 
coagulated,  this  bacillus  also  develops 
well. 

Pathogenesis. — The  discovery  of 
the  tubercle  bacillus,  its  methods  of 
cultivation  and  differential  staining, 
may  be  ranked  with  the  greatest  of 
medical  discoveries.  This  organism 
causes  in  man  and  cattle,  chiefly,  the 
disease  called  tuberculosis.  It  rarely 
attacks  the  carnivora,  but  has  been 
found  in  such  animals  when  confined. 
Swine  are  often  infected;  cats  and 
dogs  sometimes,  but  sheep,  goats,  and 
horses  seldom.  It  is  easy  to  inoculate 
guinea  pigs  or  rabbits  by  injection  or 
feeding.     The  disease  is  widespread, 


196  BACTERIA 

but  is  much  more  common  where  human  beings  are  huddled  to- 
gether in  dark,  badly  ventilated  rooms  and  shops.  In  tissues,  the 
characteristic  lesion  is  a  tubercle.  This  is  a  globular  mass,  about 
the  size  of  a  very  small  shot,  and  grayish  pearly  white.  Microscopi- 
cally, in  the  center  of  the  tubercle,  are  found  several  large  multinu- 
clear  cells,  called  giant  cells,  which  often  contain  thirty  or  more  nuclei, 
and  a  number  of  tubercle  bacilli,  the  nuclei  often  being  situated  at  one 
pole,  while  the  bacilli  are  at  the  other.  About  the  giant  cells  epithe- 
lioid cells  are  grouped,  and  about  these  leucocytes  (phagocytes)  are 
massed  in  great  numbers.  No  new  blood  vessel  formation  is  ever 
found  in  the  epithelial  cell  layers,  or  among  the  giant  cells.  Owing 
to  insufficient  blood  supply  the  center  of  the  tubercle  frequently 
undergoes  caseous  degeneration.  If  the  lesion  heals,  the  caseous 
centers  become  calcareous,  and  the  periphery  changes  into  connec- 
tive tissue.  If  the  tubercles  coalesce,  great  masses  of  caseous  tissue 
form.  If  the  latter  becomes  infected  with  other  pathogenic  bacteria 
(streptococci  and  pneumococci)  rapid  softening  occurs,  with  cavity 
formation,  etc.  Tubercles  may  develop  in  any  organ  or  tissue 
of  the  body.  The  lungs,  intestines,  peritoneum,  glands,  larynx, 
spleen,  and  bones  become  infected.  The  liver  and  pancreas  seem 
to  resist  invasion  more  than  other  organs.  Bacilli  are  rarely  found 
in  the  blood  in  tubercular  diseases.  They  may,  however,  be  found 
in  the  urine,  in  kidney,  or  bladder  tuberculosis.  Milk  from  tuber- 
culous cows,  with  infected  udders,  often  contains  bacilli,  and  is 
certainly  a  means  of  transmitting  the  disease.  Cerebro-spinal 
fluid,  in  tubercular  meningitis,  often  contains  the  bacilli.  Bacilli 
may  penetrate  mucous  membranes,  and  not  cause  any  local  lesions, 
but  infect  distant  organs.  Tuberculosis  may  be  spread  in  the 
body  in  four  ways.  Sputum  may  be  swallowed  and  infect  the 
intestines,  or  it  may  attack  the  larynx  from  the  lungs.  Infection 
may  spread  by  continuity,  by  the  lymph  stream,  or  by  the  blood. 
Ingestion  of  bacilli  may  cause  intestinal  ulceration  and  invasion 
of  the  peritoneum,  also  the  tonsils.  If  the  bacilli  reach  the  blood 
stream,  the  disease  produced  is  generally  acute  miliary  in  type. 


TUBERCLE    BACILLUS  1 97 

This  is  manifested  by  the  formation  of  fine  gray  tubercles.  In 
tuberculosis  of  the  lungs  it  is  more  than  probable  that  the  bacilh  are 
inhaled.  Local  tuberculosis  has  often  followed  skin  inoculation, 
either  by  accidental  or  intentional  trauma.  Tuberculous  mothers 
may  have  tuberculosis  of  the.  genital  tract,  and  fathers,  having 
tuberculous  testes,  discharge  bacilli  in  the  semen.  Placental  trans- 
mission of  the  bacilli  from  mother  to  child  occurs. 


Fig.  70. — Tubercle  bacilli  showing  involution  forms.     (Kolle  and  Wassermann.) 


Types  of  Tubercle  Bacilli. — It  has  been  considered  probable  by 
many  observers  that  there  are  two  types  of  bacilli,  a  human  and  a 
bovine  type.  Theobald  Smith  was  the  first  to  advance  this  theory. 
Koch  has  announced  that  the  two  types  were  totally  different,  and 
that  the  human  was  incapable  of  infecting  cattle,  and  the  bovine 
was  not  pathogenic  for  man.  In  view  of  the  fact  that  cattle  are 
frequently  tuberculous,  and  the  bacilli  are  often  found  in  the  milk, 
it  is  important  to  know  if  the  bovine  type  can  develop  in  man. 
Ravenel  has  shown  that  it  is  undoubtedly  pathogenic  for  human 
beings.  Men  have  been  infected  on  the  hands,  while  performing 
autopsies  on  tubercular  cattle,  and  their  skin  lesions  showed,  histo- 
logically, unmistakable  tubercles.     Cattle  have  been  infected  by 


igS  BACTERIA 

bacilli  of  the  human  type.     The  bovine  type  of  bacillus  differs 
from  the  human  in  the  following  ways: 

1.  It  is  much  more  pathogenic  for  guinea  pigs  and  rabbits. 

2.  It  produces  more  extensive  lesions  in  cattle. 

3.  It  is  shorter  than  the  human.  . 

4.  It  produces  more  alkali  in  acid  media. 

5.  It  is  more  readily  isolated  from  original  lesions  and  does  not 
demand  animal  juices  in  culture  media  so  emphatically. 

The  subject  of  the  infectiousness  of  bovine  tuberculosis  for 
man  has  lately  been  exhaustively  studied  by  Park  and  Krumwiede. 
Their  conclusions  are  that  bovine  tuberculosis  is  practically  a 
neglible  factor  in  adults.  It  very  rarely  causes  pulmonary  tuber- 
culosis or  phthisis,  which  disease  causes  the  vast  majority  of  deaths 
from  tuberculosis  in  man,  and  is  the  type  of  disease  responsible  for 
the  spread  of  virus  from  man  to  man.  In  children,  however,  the 
bovine  type  of  tubercle  bacillus  causes  a  marked  percentage  of 
cases  of  cervical  adenitis  leading  to  operation,  temporary  disable- 
ment, discomfort  and  disfigurement.  It  causes  a  large  percentage 
of  the  rarer  types  of  alimentary  tuberculosis  requiring  operative 
interference  or  causing  the  death  of  the  child  directly  or  as  a  con- 
tributing cause  in  other  diseases.  In  young  children  it  becomes  a 
menace  to  life  and  causes  from  6 J  to  10  percent  of  the  total  fatal- 
ities from  this  disease. 

It  is  not  always  easy  to  differentiate  the  tubercle  bacillus  from 
other  pathogenic  and  comparatively  harmless  acid-fast  bacilli. 
Among  these  are  the  B.  lepra,  the  B.  smegmatis,  and  a  number  of 
organisms  found  in  butter,  milk,  hay,  grass,  and  in  the  bhnd 
worm.  Culturally,  the  difference  is  great.  "Tuberculins"  (using 
the  term  as  a  convenience  to  describe  extracts  of  cultures),  of  the 
different  acid-fast  bacilli,  if  injected  into  animals  already  infected 
with  the  same  type  of  organism  from  which  the  extract  was  made, 
cause  the  animal  to  react  toward  the  "tuberculin."  If  a  tuber- 
cular cow  was  injected  with  a  "tubercuHn"  of  a  grass  bacillus, 
no  reaction  would  occur,  while  a  tubercle  bacillus  "tuberculin" 


TUBERCLE   BACILLUS  1 99 

would  cause  the  reaction.  This  shows  that  the  grass  bacilli  and 
the  organism  infecting  the  cow  are  not  identical.  We  are  able,  in 
this  roundabout  way,  to  differentiate  the  various  acid-fasts  (Moel- 
ler.)  By  using  carbol  fuchsin  as  a  stain,  and  a  twenty-five  percent 
solution  of  H2SO4  as  a  decolorizer,  and  after  allovdng  the  latter  to 
act  for  sixteen  hours,  it  has  been  found  that  all  of  the  "acid-fasts," 
except  the  tubercle  bacilli,  are  decolorized,  but  this  is  not  always 
reliable.  The  tubercle  bacillus  resists  this  acid  solution  seventy- 
two  hours.  By  using  a  concentrated  aqueous  solution  of  methylene 
blue  as  a  stain  for  ten  minutes,  at  room  temperature,  the  tubercle 
bacillus  is  not  colored,  while  the  smegma,  timothy-hay,  and  lepra 
bacilli  are  well  stained.  The  surest  way  to  differentiate  the  tubercle 
bacillus  from  other  acid-fast  organisms  is  by  animal  inoculations. 

For  the  discovery  of  tubercle  bacilli  in  materials  apt  to  contain 
other  acid-fasts  several  methods  are  now  employed.  The  material 
to  be  examined  may  be  stained  in  the  ordinary  manner  and  then 
decolorized  by  Pappenheim  solution  or  a  saturated  solution  of 
methylene  blue  in  absolute  alcohol.  Preparations  should  be  dried 
thoroughly  before  using  such  solutions.  For  "enriching"  in 
organisms,  the  bulk  of  material,  e.g.,  sputum,  is  suspended  in 
15  percent  antiformin  (the  proprietary  name  for  a  mixture  of 
Javelle  water  and  caustic  soda),  allowed  to  stand  in  the  incubator 
for  a  while  and  the  supension  centrifuged.  In  the  sediment  many 
more  bacilli  will  be  found  than  in  the  same  bulk  of  the  raw  specimen. 
This  antiformin  seems  to  dissolve  mucus,  tissue  and  all  bacteria 
except  tubercle  bacilli.  The  method  can  be  used  to  procure 
cultures. 

Even  with  this  method  organisms  escape  detection  in  certainly 
tuberculous  lesions.  This  is  said  to  be  due  to  non-acid  fast,  but 
gram  staining  granules.  They  are  said  to  be  found  by  a  modified 
Gram-Weigert  staining,  according  to  Much.  Such  specimens 
should  always  be  injected  into  guinea  pigs  for  corroboration. 

Immunity. — It  is  possible  to  immunize  cattle  against  virulent 
bovine  tubercle  bacilli  by  inoculating  them  previously  with  a  cul- 


200  BACTERIA 

ture  of  human  tubercle  bacilli  that  have  been  grown  for  some 
time  on  culture  media,  and  thus  attenuated.  The  new  tuberculins, 
if  injected  into  a  person  with  chronic  tuberculosis,  stimulate  the 
development  of  anti-tuberculins,  which  act  as  a  means  of  prevention 
or  defense  against  further  infection.  Thus  far  anti-tubercular 
sera  are  not  of  a  pronounced  or  certain  therapeutic  value.  By 
immunizing  horses,  Maragliano  obtained  a  serum  that  he  claims  is 
effective.  The  milk  from  immunized  cattle  is  used  as  a  diet  in 
tuberculous  patients  by  him.  The  various  tubercuHns,  some  con- 
taining endo-toxins,  or  plasmins,  in  solution,  are  capable  of  stimu- 
lating the  formation  of  agglutinins  in  the  sera  of  man  and  animals. 
Blood  from  infected  individuals  also  contains  these  bodies.  The 
agglutination  test  does  not  seem  to  be  of  great  practical  diagnostic 
value. 

BACILLUS  OF  LEPROSY. 

Mycobacterium  Lepra.     Hansen. 

Lepra  Bacillus. 

An  acid-fast  organism  resembling  the  tubercle  bacillus  morpho- 
logically when  seen  in  secretions.  The  leprosy  bacillus  from 
cultures  presents  a  pleomorphic  picture  of  short  and  long  slender, 
straight  or  slightly  bent  rods  sometimes  in  filaments  and  possessing 
deeply  staining  areas  mixed  with  unstained  ones.  It  is  shorter 
than  the  tubef  cle  bacillus,  is  non-motile,  and  probably  has  no  spores. 
In  general  it  greatly  resembles  the  tubercle  bacillus,  morphologic- 
ally and  tinctorially,  though  the  granules  are  coarser  and  farther 
apart  in  the  B.  lepra.  Certain  branched  forms  appear.  The 
morphology,  at  times,  is  Uke  the  diphtheria  bacillus.  It  stains 
by  Gram's  method,  also  by  carbol-fuchsin.  It  is  acid-fast,  but 
does  not  resist  the  action  of  acids  nearly  so  well  as  the  tubercle 
bacillus. 

Note. — ^Tubercle  bacilli  causing  avian  and  fish  tuberculosis,  and  other  acid- 
fast  bacilli  exist,  but  not  being  pathogenic  for  man,  are  not  described  here. 


RAY   FUNGUS  20I 

Cultures  have  been  made  on  serum  and  glycerine  agar,  which, 
though  resembHng  the  tubercle  bacillus,  are  more  delicate,  and  not 
so  luxuriant.  To  cultivate  the  leprosy  bits  of  tissue  are  stripped 
off  and  allowed  to  digest  with  trypsin  on  blood  serum  or  agar 
plates.  When  the  tissue  has  softened  and  the  bacilli  multiplied, 
transfers  are  made  to  serum  glycerine  media  or  those  containing 
tryptophan.  It  is  best  alkaline  in  reaction.  The  growth  is  moist 
and  pale  yellow  or  later  pink.  It  is  aerobic.  The  more  recently 
isolated  strains  grow  very  slowly.  Variations  in  the  media  produce 
various  grades  of  pigmentations.  Apparently  leprosy  bacilli  cannot 
break  up  complex  protein  molecules. 

Pathogenesis. — It  is  highly  pathogenic  for  man  and  monkeys, 
producing  in  the  former  a  slow  chronic  disease,  which  is,  probably, 
transmitted  by  more  or  less  intimate  personal  contact.  The  bacillus 
is  seen  in  enormous  numbers  in  lepra  cells  and  elsewhere  in  diseased 
tissues  and  has  been  found  in  the  blood.  The  lepra  cells  are  large 
and  vacuolated,  and  literally  crammed  full  to  bursting  with  bacilli. 
In  general  the  leprous  lesion  resembles  a  tubercle,  as  it  consists  of 
giant  cells,  epithelial,  and  round  cells. 

Immunity. — There  is  very  Uttle  accurate  knowlege  as  to  immu- 
nity against  this  organism;  of  late  bacterins  have  been  tried  with 
some  success  it  is  claimed. 

RAY  FUNGUS. 

Actinomyces  Bovis. 

Ray  Fungus. 

Morphology  and  Stains. — This  organism  is  called  the  ray  fungus 
because  of  the  stellate  arrangement  of  its  threads  in  the  colonies 
found  in  tissues.  It  is  of  a  more  complex  structure  than  the  bac- 
teria hitherto  described.  There  are  three  elements  found  in  every 
colony:     i.  Long  thread  which  may  be  branched  or  unbranched. 

2.  Threads  that  are  clubbed,  which  may,  or  may  not,  be  branched. 

3.  Spore-like  bodies  contained  within  the  thread,  from  the  ends  of 


202 


BACTERIA 


which  they  are  discharged.  The  colonies  in  tissues  are  often  i  mm. 
in  diameter,  and  made  up  of  many  clubbed-shaped  threads  radi- 
ally situated.  Through  the  periphery  and  extending  beyond  are 
other  unclubbed  threads,  while  scattered  throughout  the  colony 
and  beyond  it,  and  in  the  threads,  may  be  seen  many  spore-like 
bodies.  The  threads  and  spores  stain  by  Gram's  method,  while 
the  clubs  do  not.  Basic  stains  also  color  all  the  elements.  The 
spores  do  not  stain  like  bacterial  endo-spores. 


Fig.  71. — Actinomyces  bo  vis.     (Williams.) 

Vital  Requirements. — It  is  a  facultative  aerobe,  and  grows  best 
in  the  presence  of  air,  at  37°  C.  Resists  drying  for  a  long  time, 
and  its  thermal  death-point  is  80°  C.  after  fifteen  minutes  exposure. 
Chemical  Activities. — Slowly  liquefies  gelatine,  does  not  curdle 
milk;  and  produces  a  mouldy  odor.  No  gas  or  acids  are  formed, 
nor  is  HgS  developed. 

Habitat. — It  has  been  found  in  straw  and  hay,  but  never  in  a 
healthy  body. 


FARCIN  DU  BOEUF  203 

Cultures. — On  gelatine  plates  it  produces  yellowish-gray  colo- 
nies that  are  very  small.  These  grow  into  the  gelatine,  slowly  lique- 
fying it.  The  colonies  are  very  tough  and  fibrous.  In  agar  tubes  it 
grows  very  slowly,  the  first  growth  being  hke  dew-drops;  later  these 
enlarge,  turning  yellow,  and  finally  brown.  The  culture  grows 
down  into  the  agar,  and  the  medium  darkens.  Old  cultures  are  dark 
and  crumbly  looking,  adhere  firmly  to  the  agar,  and  have  a  downy 
dust-Hke  covering.  On  blood  serum  the  colonies  appear  as  dew- 
drops,  which  later  become  brownish,  then,  yellowish-orange,  or 
brick-red.  In  bouillon  the  growth  is  at  the  bottom  in  ball-Hke 
masses,  that  firmly  cohere.  Clubs  do  not  form  in  this  medium. 
The  supernatant  bouillon  is  clear,  with  no  surface  growth.  In 
milk  it  produces  no  chemical  change.  On  potato  it  grows  in 
knot-like  colonies. 

Pathogenesis. — Causes  in  cattle  the  disease  known  as  "lumpy 
jaw."  The  fungus  reaches  the  jaw  from  the  teeth  and  gums,  the 
latter  first  being  injured  by  sharp  spines  in  the  food.  In  man,  the 
internal  organs,  lungs,  intestines,  and,  rarely,  the  brain  become 
infected.  The  liver  often  is  abscessed.  In  both  cattle  and  man 
universal  actinomycosis  sometimes  occurs.  It  is  hard  to  inoculate 
laboratory  animals  with  the  disease,  though  Wright  succeeded  in 
so  doing.  The  lesions  produced  are  rather  massive  at  times;  the 
nidus  is  often  surrounded  by  enormous  numbers  of  polynuclear 
leucocytes,  which,  no  doubt,  play  a  defensive  role  in  the  tissues. 
The  disease  is  often  fatal  to  cattle  and  to  man. 

FARCIN  DU  BOEUF. 

Actinomyces  Farcinicus. 

Bacillus  du  farcin  du  Boeuf.     No  card. 

Morphology  and  Stains. — Segmented  threads  with  true  branch- 
ing, short  and  knotty,  or  long  and  delicate.  Contains  spores,  is  not 
motile,  and  has  no  flagella.  It  stains  with  all  the  ordinary  aniline 
dyes,  and  by  Weigert-Gram  method.  Ziehl's  method  stains  it  well. 
It  is  often  seen  as  tangled  masses  of  threads. 


204  BACTERIA 

Vital  Requirements. — Grows  well  at  room  temperature,  and  in 
the  incubator.  Nocard  kept  a  culture  at  40°  C.  for  four  months 
and  it  was  still  virulent. 

Cultures. — It  thrives  well  on  all  culture  media.  On  bouillon  the 
growth  is  colorless,  and  in  masses  that  float  and  then  sink;  or  in  a 
fenestrated  pellicle  on  the  top.  On  Agar. — It  appears  in  discrete 
litde  roundish  yellowish-white  masses  that  resemble  lichens.  On 
blood  serum  its  growth  is  like  that  on  agar,  only  less  luxuriant. 
On  potato  it  is  scaly,  wrinkled,  yellowish  and  dry.  In  milk  the 
organism  flourishes,  without  curdling  the  milk  or  altering  its 
reaction. 

Pathogenesis. — This  organism  is  pathogenic  for  all  the  laboratory 
animals.  Sheep,  dogs,  wild  rabbits,  horses,  asses,  and  men  are 
immune.  It  produces  an  abscess,  in  those  animals  for  which  it  is 
pathogenic,  that  discharges,  with  subsequent  induration,  ulceration, 
and  sloughing.     The  disease  in  cattle  resembles  glanders. 

If  injected  into  the  blood,  miliary  tubercles  are  found  that 
resemble  tuberculosis. 

ACTINOMYCES  MADURA. 

Actinomyces  Madura. 

Streptolhrix  MadurcB,  Vincent. 

Morphology  and  Stains. — A  non-motile,  non- flagellated  organ- 
ism said  to  have  spores.  Its  growth  resembles  that  of  Actinomyces 
bovis.  It  consists  of  long  threads  that  are  clubbed.  These  stain 
by  all  the  basic  aniline  dyes  and  by  Gram's  method. 

Vital  Requirements. — It  is  a  facultative  aerobe.  The  thermal 
death-point  for  the  spores  is  85°  C.  for  three  minutes,  and  75°  C. 
for  five  minutes.  Vegetative  thread  forms  die  at  60°  C.  Grows 
best  at  37°  C.,  and  scantily  at  room  temperature. 

Cultures. — Generates  upon  all  culture  media.  In  Bouillon. — It 
appears  in  httle  clumps  which  cHng  to  the  glass,  and  are  bright  red 
in  color,  eventually  they  sink  to  the  bottom  in  pale  masses.  In 
Gelatine. — It  grows  sparingly  in  clumps,  slowly  liquefying  the 


STREPTOTHRIX 


205 


medium.  Upon  Agar. — It  forms  shiny  round  colonies,  that  are  first 
devoid  of  color,  then  become  deep  red.  They  resemble  an  umbili- 
cated  vaccine  vesicle  and  adhere  tightly  to  the  agar.  In  Milk. — It 
grows  without  coagulating  the  medium.  On  Potato. — The  culture 
is  very  slow,  and  without  chromogenesis.  Old  colonies  are  powdery, 
due  to  spores. 


Fig.  72. — Streptothrix  hominis.     (Kolle  and  Wassermann.) 

Pathogenesis. — In  man  it  produces  madura  foot,  an  affection 
characterized  by  induration,  ulceration,  and  fistulas  formation  with 
pus. 

STREPTOTHRIX  (Eppinger). 

The  genus  of  truly  branching  mycelium- forming  higher  bacteria 
(see  page  3).  The  same  genus  includes  the  actinomyces.  Kruse 
has  described  nineteen  different  members  of  the  streptothrix,  some 
pathogenic  to  man  and  animals. 

Lately  a  number  of  cases  of  streptothrix  (Streptothrix  Hominis) 
infection  in  man  have  been  reported.  The  disease,  in  general, 
resembles  phthisis.  In  the  pus,  sputum,  and  stained  sections  xyi 
these  cases,  strep  to  thricial  threads  have  been  found.     (Fig.  72.) 


2b6  BACTERIA 

Morphology  and  Stains. — Threads  are  thick  and  short,  or  long 
and  slender,  depending  upon  the  medium  on  which  they  grow.  In 
bouillon  the  threads  are  thin  and  long,  on  blood  serum,  short  and 
thick.  When  stained  there  is  distinct  beading  and  fragmentation 
of  the  protoplasm. 


Fig.  73. — Streptothrix  Candida.     (Kolle  and  Wassermann.) 

There  is  true  branching  of  an  irregular  type,  which  is  best  seen 
in  Hquid  media.  These  threads  often  produce  spores  on  culture 
media.  The  threads  often  disappear  in  old  cultures,  leaving  only 
the  spores,  which  stain  with  carbol-fuchsin  and  do  not  decolorize. 
The  threads  stain  by  Gram's  method,  and  Gram-Weigert  method. 
The  threads  are  not  acid-fast. 

Vital  Characteristics. — These  organisms  live  for  years  in  cul- 
ture media  after  it  is  dry.  Spores  resist  dry  heat  at  60°  0.-70°  C. 
for  an  hour;  moist  heat,  60°  C.  however,  kills  them  after  an  hour. 
It  is  a  strict  aerobe. 

Cultures. — On  Loffler's  blood  serum,  according  to  Tutde,  this 
organism  grows  slowly  in  whitish  colonies,  which  finally  become 
yellow.  The  adult  colonies  adhere  to  the  serum.  On  Agar  it 
grows  rapidly  and  characteristically.     The  colonies  are  yellowish- 


OIDIOMYCOSIS  207 

white  and  adhere  to  the  agar.  In  Bouillon. — It  develops  slowly 
on  the  surface  of  the  medium.  Fluffy  tufts,  or  balls,  are  formed, 
that  sink  to  the  bottom  of  the  tube.     The  growth  is  whitish. 

Pathogenesis. — For  rabbits  and  guinea  pigs  this  organism  is 
pathogenic,  producing  abscesses,  tubercles,  induration,  etc.  It  is 
a  pus  forming  organism. 

In  man,  the  disease  picture  is  like  that  of  tuberculosis.  It  causes 
abscesses,  adenitis,  indurations  of  the  skin,  endocarditis,  and  pleuri- 
tic inflammation.  Many  grayish  tubercles  were  found  that  resem- 
bled the  lesions  produced  by  the  tubercle  bacillus.  Cavity  for- 
mation has  been  described. 

This  organism  acts  as  a  secondary  infecting  agent  in  tuberculosis 
of  the  lungs.     Tuttie  reviews  twelve  cases,  all  of  which  were  fatal. 

In  examining  sputum  from  tubercular  cases,  in  which  the  typical 
bacilli  are  not  found,  it  is  well  to  look  for  the  streptothrix  by  staining 
with  Gram's  stain. 

Leptothrix  Buccalis. — Long  unbranched  threads  that  grow  in 
the  walls  of  the  pharynx,  causing  very  sore  throat.  This  organism 
has  not  been  cultivated,  hence,  very  little  is  known  of  it.  It  is  not  a 
member  of  the  actinomyces,  because  it  is  not  branched,  nor  is  it  a 
streptothrix  for  the  same  reason. 

Leptothrix  Vaginalis. — Is  another  variety  that  has  been  found 
growing  in  the  vagina.  Nothing  is  known  of  its  pathogenicity,  nor 
of  its  cultural  properties. 

BLASTOMYCOSIS. 
OIDIOMYCOSIS. 

Oidium  Albicans.  Thrush,  Soor. — A  member  of  the  higher 
order  of  the  fungi.  This  organism  resembles  both  a  yeast  and  a 
mould,  because  it  exhibits  characteristics  that  are  common  to 
both  of  these  two  forms.  It  exhibits  budding  yeast  cells  and 
budding  myceha.  The  yeast  cell  is  6jx  long  and  i/£  wide,  but  the 
cells  vary  very  much  in  length  and  width. 


2o8  BACTERIA 

It  Stains  well  in  tissues  and  cultures  by  Gram's  method,  and  by 
the  ordinary  basic  stains.  It  may  be  cultivated  on  bouillon,  blood 
serum,  agar,  potato,  etc.,  and  it  is  rather  indifferent  to  the  reaction 
of  the  media.  It  grows  best  if  sugars  are  present.  It  is,  however, 
very  susceptible  to  such  antiseptics  as  phenol,  salicyHc  acid,  sub- 
limate, etc. 


Fig.  74. — Thrush  fungus.     (Kolle  and  Wassermann.) 

Pathogenesis. — Causes  in  man  a  condition  known  as  oidio- 
mycosis, and  in  young  children  a  very  troublesome  stomatitis,  which, 
if  the  child  is  weak  and  illy  nourished,  may  result  seriously.  It  may 
cause  metastatic  abscesses  in  the  brain,  spleen,  and  kidneys,  or 
nodules  in  the  lungs.  This  organism  may  penetrate  mucous  tissues, 
and  fill  the  lumen  of  vessels  (Virchow).  By  repeated  injections  of 
cultures  into  rabbits  anti-oidium  serum  may  be  prepared.  This 
serum  exercises  a  bacteriolytic  and  an  agglutinative  action  on  the 
oidium  which  normal  serum  does  not  have. 

Oidium  Coccidioides,  Ophiils.  Saccharomyces  Busse.  (Blas- 
tomycetes). — In  and  near  Chicago  there  have  appeared  parasitic 
inflammations  of  the  skin  that  have  been  termed  blastomycetic 
dermatitis.     From   the   lesions   of   this   disease   fungi   have   been 


MOULDS  209 

cultivated  which  resemble  closely  the  blastomycetes,  but  Ricketts 
and  Ophuls  have  placed  this  organism  in  the  oidium  genus.  Not 
only  does  it  cause  an  infectious  dermatitis,  but  it  may  invade  the 
deeper  tissues  and  organs.  The  lungs  may  be  primarily  invaded, 
setting  up  in  them  an  oidiomycosis  that  resembles  or  imitates  in  its 
general  appearance  pulmonary  tuberculosis.  The  oidium  may  be 
detected  in  the  sputum,  and  exhibits  budding.  It  is  easily  stained. 
The  diseases  and  organism  described  by  Busse  and  Gilchrist  are 
probably  closely  related  to  Ophuls  pictures.  There  seem  to  be 
several  species  of  pathogenic  yeasts  capable  of  a  variety  of  influ- 
ences. It  is  better  to  classify  them  all  under  Saccharomyces,  as 
there  are  no  fundamental  differences  between  Ophiils  oidia  and 
Busse' s  yeast.  The  character  of  the  lesions  depends  upon  the  point 
of  entry.  The  yeast  in  the  tissue  presents  doubly  contoured, 
highly  refractive  discs  from  which  buds  and  short  mycelia  grow. 
These  so-called  hyphae  may  intertwine.  They  may  be  obtained  in 
culture  by  injecting  a  guinea  pig  and  culturing  out.  They  grow  in 
a  white,  fluffy  mass  on  agar  and  gelatine. 

MOULDS  OR  HYPHOMYCETES 

Aspergillus  Niger,  A.  Fumigatus,  and  A.  Flavus. — A  poly- 
cellular  mycelial  organism  which  produces  spores  and  branched 
threads,  that  are  variously  named  from  the  macroscopic  ap- 
pearances of  the  growth.  All  thrive  well  as  37°  C.  and  may  be 
cultivated  on  the  usual  culture  media.  In  man,  the  external  auditory 
meatus  is  often  infected  with  these  orgnaisms,  causing  a  trouble- 
some disease.  They  may  infect  the  lungs  of  weak  anemic  subjects 
with  wasting  diseases,  and  may  be  pathogenic  for  cattle,  horses, 
and  birds. 

The  author  has  found  that  the  young  hyphae,  the  sporangia, 

and  spores  of  some  of  these  hyphomycetes  (moulds)  if  treated  with 

hot  or  boihng  alkaline  solution  of  copper  sulphate  are  stained  by  the 

copper,  which  has  an  affinity  for  them,  and  appear  a  light  lilac  blue 

14 


2IO  BACTERIA 

under  the  microscope.  If  treated  with  a  solution  of  ferro  cyanide 
of  potash  and  acetic  acid,  these  stained  parts  turn  a  dark  brown, 
showing  that  there  is  an  actual  absorption  or  perhaps  chemical 
union  of  the  protoplasm  of  the  mould  with  the  copper.  Some 
moulds  are  stained  a  deep  blue,  and  are  visible  to  the  naked  eye  in 
test-tubes,  after  treatment  with  the  boiling  alkaUne  copper  others 
are  colored  a  bright  yellow.  Some  moulds  and  bacteria  have  the 
power  of  reducing  copper  in  Fehhng's  solution. 

Diseases  due  to  these  forms  are  practically  confined  to  the  skin 
although  extremely  rare  cases  of  dissemination  are  on  record. 

Ringworm  of  all  kinds  is  due  to  the  mould  Trichophyton  either  of 
the  species  megalosporon  or  microsporon.  The  spores  of  the 
former  are  7-8//,  of  the  latter  2-3//.  They  grow  readily  as  dis- 
crete mammillated  fluffy  colonies.  They  consist  under  the  micro- 
scope of  slender  septate  hyphae. 

Favus  is  due  to  the  mould  Achorion  Schoenleinii.  This  fungus 
gives  off  hyphae  with  knob-like  reproductive  organs.  Spores  are 
oval  3-8/^X3-4/^.  This  fungus  grows  as  a  "scutulum"  on  the 
skin  eruption.  It  can  be  cultivated  on  sugar  agar,  as  a  waxy,  or 
downy  yellow  or  white  round  plate  with  a  central  mammillation. 

Pityriasis  versicolor  is  due  to  the  mould  Microsporon  furfur. 
It  is  similar  to  the  Trichophyta,  but  only  invades  the  supeificial 
layers  of  the  skin. 


CHAPTER  IX. 

ANIMAL  PARASITES. 

While  numerous  diseases  are  caused  by  vegetable  parasites,  such 
as  bacteria  and  moulds,  there  are  others  in  which  the  etiological  role 
is  played  by  minute  microscopic  organisms  of  the  animal  kingdom. 
There  are  also  infectious  diseases  that  are  supposedly  caused  by 
animal  parasites,  and  yet,  the  exact  knowledge  that  they  are  the 
cause  is  lacking.  Not  all  of  the  pathogens  of  the  animal  kingdom 
will  fulfil  Koch's  postulates  but  their  number  is  increasing. 
Within  the  past  few  years  it  has  been  found  possible  to  cultivate 
Trypanosomata,  spirochaetae,  amoebae,  and  hemosporidia  with 
completion  of  Koch's  postulates  in  the  first  two. 

In  general,  it  may  be  said  of  animal  parasites,  particularly  those 
belonging  to  the  protozoa,  that  an  intermediate  host,  such  as  a 
suctorial  insect,  is  necessary  for  the  transmission  of  the  organism 
to  man  or  animal.  This  is  called  alternate  generation  and  is  a  very 
characteristic  feature. 

The  protozoa,  as  parasites  in  man,  are  the  cause  of  several  well- 
known  diseases,  namely: — Dysentery,  malaria,  sleeping-sickness, 
and  coccidiosis.  In  hydrophobia,  scarlet  fever,  and  small-pox 
certain  peculiar  bodies  are  constantly  found  that  resemble  protozoa, 
but  since  it  is  not  known  whether  they  are  animal  bodies  at  all,  they 
cannot  be  classed  as  protozoa,  though  they  will  be  described  as  such. 

PROTOZOA. 

The  protozoa  of  importance  as  disease  producers  are  to  be  found 
in  the  classes,  orders  and  families  given  as  follows. 

211 


212  ANIMAL  PARASITES 

Protozoa. 

Sarcodina. 

Rhizopoda. 

Gymnamoebida — Amoebae. 
Mastigophora. 
^     Flagellata. 

Monadida,     Cercomonas,    Trypansosoma,    Poly- 
mastigida,  Trichomonas. 
Some  authors  separate  a  family  Spirochaetidae  to  include 
Spirochaeta  and  Treponema. 
Sporozoa. 

Gregarinida — gregarines. 
Coccidia — coccidia. 
Hemosporidia. 

Plasmodium — malaria. 
Infusoria. 
Ciliata. 

Heterotrichida— Balantidium. 

The  protozoa  are  always,  in  every  stage  of  development,  primi- 
tive unicellular  bodies.  They  consist  essentially  of  a  cell-body  or 
sarcode,  a  nucleus,  and  a  nucleolus.  All  of  the  vital  functions  of  the 
cell  are  carried  out  by  the  cell-body,  the  protoplasm  of  which  digests 
and  assimilates  food.  Particular  parts  of  the  protoplasm  have 
special  functions,  these  parts  are  called  organelles.  The  living 
protoplasm  is  finely  granular,  is  viscid,  and  exhibits  a  distinct 
movement.  The  motility  of  protozoa  is  suppHed  variously.  In 
the  Rhizopoda  progression  takes  place  by  pseudopods  or  false  feet, 
a  phenomenon  in  which  a  section  of  the  cell  wall  and  protoplasm 
are  extended  like  a  bud.  Into  this  the  latter  then  flows  with  a 
shrinkage  of  the  main  body.  At  last  the  pseudopod  is  large  enough 
to  hold  all  the  protoplasm  and  the  former  place  of  the  protozoon  is 
vacated  for  the  new.  Motility  is  also  supplied  by  the  lashing  or 
vibratory  action  of  flagella  or  the  fine  vibration  of  the  circum- 


AMCEBA  D YSENTERIiE  2 1 3 

ferential  cilia.  In  others  a  special  muscular  segment  of  the  body 
may  exist.  The  suctorial  tubes  act  also  for  motion  at  times.  In 
most  protozoa  two  layers  can  be  seen — the  ectosarc,  and  endosarc. 
The  ectosarc  originates  the  movement,  is  concerned  in  the  ingestion 
and  excretion  of  food,  and  the  respiration.  The  endosarc,  which 
circulates  slowly,  is  mainly  for  digestive  purposes.  In  it  are  fer- 
ments, crystals,  food  particles  (seen  in  the  food  vacuoles),  oil 
globules,  gas,  and  pigment  granules. 

Flagella  and  suctorial  tubes — in  protozoa  that  have  them — ^belong 
to  the  ectosarc.  Skeletal  tissues,  shells,  etc.,  also  belong  to  this 
layer. 

The  food  consists  of  bacteria,  smaller  animals,  algae,  and  animal 
waste. 

Propagation  is  effected  by  direct  cell  division,  beginning  in  the 
nucleus,  by  cell  budding  or  by  a  complicated  course  of  sporulation 
which  may  be  sexual  or  asexual.  Sometimes  division,  or  budding, 
occurs  rapidly  without  the  segments  separating,  leading  to  the 
formation  of  protozoa  colonies,  or  swarm  spores. 

In  the  case  of  the  malarial  plasmodia,  asexual  development, 
{schizogony)  takes  place  in  man's  blood,  while  the  sexual  develop- 
ment {sporogony)  takes  place  in  the  mosquito.  Protozoa  are  found 
in  salt  and  fresh  water,  in  damp  places,  and  in  animals  as  parasites. 

Since  the  zoological  classification  has  been  given  and  may  be  used 
for  reference  to  larger  works,  the  various  pathogenic  protozoa  are 
given  separately  without  direct  reference  to  their  systematic 
classification. 

There  are  but  two  Rhizopods  that  are  parasitic  and  pathogenic  to 
man.     The  only  one  of  these  of  any  import  is  the  Amoeba. 

AMCEBA  DYSENTERIiE  OR  ENTOMCEBA 

HISTOLYTICA. 

• 

This  is  a  pear-shaped  roundish  body  from  .008  to  .05  mm.  in 
diameter.  The  ectosarc  is  easily  discernible  in  the  pseudopodia, 
but  not  in  the  round  quiescent  cell.     In  the  endosarc,  which  is 


214 


ANIMAL   PARASITES 


®C   N 

©  ^S^ 

%^-'  ^  m 

granular,  vacuoles  are  easily  seen;  so  are  fragments  of  food,  red 
and  white  blood  cells,  bacteria,  epithelial  cells,  and  fecal  matter. 
The  pseudopodia  are  broad  and  lobose;  one  or  two  are  protruded 
at  a  time.  The  motion  of  the  organism  depends  upon  the  reaction 
of  the  media,  and  the  temperature.  The 
vacuoles  and  nucleus  are  always  present. 
Propagation  generally  takes  place  by 
binary  division,  the  process  beginning  in 
the  nucleus.  When  irritated,  the  amoeba 
at  once  assumes  a  spherical  form,  the 
pseudopodia  being  withdrawn. 

Pathogenesis. — Amoeba   dysenteriae  is 
always  pathogenic.     It  is  now  considered 
Pig.  75.— Amoeba  dysen-   the  cause  of  the  protozoal  form  of  dysen- 
teriae    (Greene's    Medical  tery.     So    far   as    known  this   particular 
Diagnosis.)  .   ,  .  ,  ,       .        ,        . 

variety   exists    only   in   the   intestines    of 

affected  persons.  Lesions  similar  to  those  of  human  dysentery  have 
been  produced  in  monkeys,  dogs  and  cats,  and  the  amoebae  recovered 
from  them.  Cultures  consisting  only  of  amoebae  have  been  ob- 
tained by  special  technique,  but  a  so-called  pure  mixed  growth  of 
colon  bacilli  and  amoebae  is  cultivated  with  little 
difficulty.  In  the  lower  gut  of  man  and  cats,  in 
dysentery  cases,  encysted  amoebae  are  often 
found.  They  have  been  seen  in  the  liver  (in 
old  cases),  also  in  the  lungs  and  sputum.  In 
over'  500  cases  of  dysentery  the  amoeba  was 
present  in  every  instance. 

Cats   have   been  infected  by  pus  from  liver 
abscesses    devoid  of  bacteria    (Kartulis). 


Fig.  76.- 


-Ameboid 
The  motion.       (Greene's 
r         ,.^.  ^  .       1  1         Medical  Diagnosis.) 

unne,   in   cases   of  cystitis,  contained  amoebae, 

and  it  is  believed  to  be  the  cause  of  the  disease  irj  some  rare 
instances.  In  dysentery  the  amoebae  are  the  cause  of  the  necrosis 
and  ulceration,  as  they  frequently  become  encysted  in  the  submu- 
cous tissues.     From  the  Entomceba  coHc  the  dysenteric  amoeba  is 


FLAGELLATA  21 5 

differentiated  by  the  fact  that  it  is  larger,  coarser  in  structure,  and 
takes  up  red  blood  cells,  which  the  former  does  not.  Differentia- 
tion by  Wright's  stain  Entomoehacoli  ectoplasm  lighj;  blue,  endoplasm 
dark  blue,  nucleus  red.  Ent.  histolytica  ectoplasm  dark  blue,  ento- 
plasm  light  blue,  nucleus  pale  red  or  pink. 

In  stools  (from  dysenteric  cases)  over  a  day  old,  amoebae  are  not 
often  found,  as  they  undergo  a  rapid  disintegration  outside  the  body. 

Amoebae  are  cultivated  upon  stiff  agar  in  company  with  bacteria. 
If  a  colony  can  be  obtained  free  of  bacteria,  development  will  con- 
tinue on  agar  smeared  with  organ  extracts.  The  addition  of  dead 
bacteria  to  culture  media  seems  favorable  to  their  development. 
The  poisin  is  not  known.  The  free  amoebae  in  the  colon  are  easily 
killed,  but  when  encysted  are  more  resistant.  Quinine  is  fatal  to 
cultures  in  10  minutes  in  strength  of  1-2500.  FormaUn  is  not 
practicable. 

The  two  varieties  closely  resembling  Ent.  histolytica  sue  Entamoeba 
coli  and  Ent,  tetragena.  They  vary  in  finer  morphological  details, 
in  their  reproduction  and  their  pathogenic  properties.  These  two 
varieties  are  not  supposed  to  be  pathogenic  for  man.  According  to 
some  authorities  sulphur  in  some  form  is  necessary  for  the  growth  of 
amoebae. 

FLAGELLATA. 

The  flagellata  derive  their  name  from  the  fact  that  all  are  pos- 
sessed, at  some  time  in  their  existence,  of  flagella,  which  are  not  only 
organs  of  locomotion,  but  serve  to  apprehend 
food. 


*K 


The  principal  members  of  this  class  of  in- 
terest from  a  pathological  view-point,  are  the 
trypanosomes.     Trypanosoma     gambiense,    Fig.  77.— -Flagellata. 
transmitted  by  the  tsetse-fly  Glossina  palpaUs  i^iagnosi^.)  ^ 
pathogenic   for   man    (see   page    218).     The 
Trypanosoma  brucei,  which  causes  the  tsetse-fly  disease  (nagana) 
in  horses  and  cattle,  is  transmitted  to  cattle  by  the  bite  of  the 


2l6 


ANIMAL   PARASITES 


tsetse- fly,  glossinia  morsitans.  It  can  be  grown  on  blood  agar 
(Novy). 

Trypanosoma  evansi  causes  surra,  a  disease  of  horses  in  Cetitral 
Asia. 

Trypanosoma  equiperdum  causes  a  sexual  disease  in  stallions 
and  mares  called  dourine ;  this  is  akin  to  syphilis  in  man. 

Trypanosoma  lewisi  of  rats  is  transmitted  from  animal  to 
animal  by  means  of  fleas. 


Fig.  78. — Typanosome  in  rats'  blood.     (Williams.) 

Trypanosoma  noctuae. — A  parasite  of  the  Httle  owl,  which  is 
introduced  into  the  bird  through  the  bite  of  the  mosquito  Culex 
pipiens. 

Trypanosomas  are  elongated  fusiform  bodies  pointed  at  both 
ends,  provided  by  a  fin  fold,  or  undulating  membrane,  running 
along  the  dorsal  edge  and  forming  frill-Hke  folds  which  terminate 
in  a  whip-like  extremity  or  flagellum. 


TRYPANOSOMA   GAMBIENSE  217 

A  large  nucleus  is  always  seen,  also  a  centrosome,  a  small  chro- 
matic mass — likewise  called  a  blepharophast — near  one  pole. 

The  flagellum  is  at  the  anterior  extremity;  the  short  pointed  end 
is  the  posterior  extremity.  Cell  division  begins  in  the  nucleus,  the 
cell  dividing  longitudinally,  the  centrosome,  flagellum,  and  the 
protoplasm  dividing  last.  Trypanosomes  frequently  appear  in 
clumps  with  the  ends  united,  resembling  a  wheel. 

The  trypanosomes  exist  in  two  hosts — one  a  suctorial  insect — and 
have  a  sexual  and  an  asexual  existence  (alternate  generation). 

In  an  infected  owl  the  organism  has  been  observed  cUnging  fast 
to  the  red  cells,  absorbing  nutriment  during  the  day,  while  at  night 
it  swims  about  freely  in  the  plasma. 

In  owl's  blood  the  trypanosome  assumes  asexual  forms,  called 
macro  gametes.  These  macrogametes  penetrate  the  erythrocytes, 
accumulating  the  remnants  of  the  red  cells  in  the  protoplasm.  The 
nucleus  of  the  trypanosome  may  be  seen  in  the  interior  of  the  pro- 
toplasm. The  microgametocytes  arise  from  the  asexual  forms  and 
when  mature,  give  rise  to  eight  microgametes. 

TRYPANOSOMA  GAMBIENSE. 

Castellani  found  that  this  trypanosome  is  the  cause  of  sleeping 
disease  among  the  natives  of  South  Africa.  The  organism  has 
been  found  in  the  cerebro-spinal  fluid — in  cases  of  sleeping-sickness 
— quite  uniformly.  They  have  also  been  found  in  the  blood.  The 
disease  has  a  long  period  of  incubation  (months) ,  runs  a  long  course 
usually,  and,  at  its  full  development,  it  is  a  meningo-encephalo- 
myehtis.  This  is  characterized  by  hebetude,  somnolence,  and 
coma.  These  symptoms  are  accompanied  by  disturbance  of  the 
motor  apparatus,  oedema,  irregular  temperature,  rapid  pulse, 
emaciation,  skin  eruptions,  and  death  in  coma.  In  these  cases  the 
parasites  may  be  seen  in  the  blood  slowly  winding  their  way  through 
the  corpuscles.  The  pathogenic  action  is  due  no  doubt  to  some 
toxin  elaborated. 


2l8  ANIMAL   PARASITES 

The  disease  is  transmitted  from  man  to  man  by  the  tsetse-fly 
(Glossina  palpalis).  In  the  fly  it  exists  as  a  true  parasite  in  a  host, 
and  not  merely  passively.  It  becomes  infective  within  three  days 
of  biting  and  remains  so  for  four  weeks. 

The  disease  does  not  depend  upon  the  age,  sex  of  the  individual, 
nor  upon  drinking  water,  food,  seasons,  etc. 

The  organism  may  be  stained  by  the  ordinary  blood  stains,  mix- 
tures such  as  Leishman's,  Romano wsky's,  etc.,  the  nucleus, 
centrosome  and  flagella,  staining  deepest.  Thus  far  the  T.  gam- 
biense  has  not  been  cultivated  in  artificial  media. 


Fig.  79. — Trypanosomes;  showing  ordinary  structural  appearance  on  left;  in 
middle  a  trypanosome  undergoing  division;  on  the  right  an  agglutinated  group. 
(Tyson's  Practice.) 

Novy  has  succeeded  in  growing  the  T.  lewisii  and  T.  brucei  on 
agar  mixed  with  defibrinated  rabbit's  blood.  These  are  the  first 
animal  parasites  to  be  cultivated  artificially. 

Trypanosomiasis  of  South  America  is  not  unlike  sleeping  sickness 
of  Africa.  It  is  caused  by  Tr.  cruzi,  a  parasite  of  8  spores,  develop- 
ing in  organs,  serum  or  red  cells.  It  is  transmitted  by  Conorrhinus 
megistus,  a  large  insect. 

In  Dum  Dum  fever  or  Kala  Azar,  a  disease  occurring  in  India, 
curious  bodies,  called  Leishman-Donovan  bodies  have  been  found. 
These  resemble  the  malarial  plasmodia  roughly,  and  if  cultivated 
on  blood  agar  elongated  herpetomas-like  bodies  without  undulating 
membranes    will    develop.     These    bodies   are   evidently  in   the 


TREPONEMA   PALLIDUM  219 

halteridium  stage  of  trypanosome  existence.  They  are  to  be  found 
in  the  juice  obtained  by  splenic  puncture.  On  the  rare  occasions 
they  have  been  met  in  the  blood  they  were  within  a  leucocyte. 
The  transmission  is  unknown. 

TREPONEMA  PALLIDUM  (Schaudinn). 

(Spirochaeta  Pallida.) 

Treponema  Pallidum. — There  has  been  some  discussion  as  to 
the  proper  classification  but  now  this  organism  is  usually  placed 
among  the  Flagellata,  genus  Treponema,  as  it  does  not  possess  an 
undulating  membrane,  is  flagellated,  is  of  stiff  and  regular  shape, 
and  multipHes  by  longitudinal  division. 

Morphology  and  Stains. — This  organism  is  extremely  delicate 
in  structure,  from  4  to  14/^  in  length  and  about  .3/^  in  width;  has 
from  3  to  12  turns  or  bends,  and  its  ends  are  delicately  pointed. 
Its  curves  form  a  large  arc  of  a  small  circle.  The  Sp.  refringens 
curves  form  a  small  arc,  frequently  irregular,  of  a  larger  circle. 
It  multiplies  by  both  transverse  and  longitudinal  division.  As  this 
organism  is  stained  vdth  difficulty  it  requires  a  special  one,  that  of 
Giemsa  yielding  the  best  results.  Aniline  gentian  violet,  Romano w- 
sky's,  and  Leishman's  stains  also  color  it.  It  may  be  stained  in 
tissues  by  silver  and  pyrogallic  acid  methods. 

Habitat. — It  has  not  been  found  in  tissues  of  normal  persons,  or 
those  ill  with  carcinoma,  tuberculosis,  etc.,  but  only  in  the  tissues 
of  individuals  suffering  with  syphilis.     It  is  a  strict  parasite. 

Vitality. — Nothing  is  known  of  its  ability  to  withstand  the  action 
of  chemicals,  Hght,  heat,  or  other  deleterious  agencies.  Glycerine 
destroys  its  motility. 

The  Treponema  pallidum  has  now  fulfilled  the  postulates  of 
Koch.  It  can  be  cultivated  from  human  lesions  (with  some 
difficulty  to  be  sure),  it  can  be  implanted  in  animals  (monkeys  and 
rabbits)  and  there  reproduce  syphilitic  lesions;  and  it  can  be 
recultivated  from  them.     In  these  experimental  diseases  it  retains 


220 


ANIMAL  PARASITES 


the  proper  morphology.  According  to  Noguchi  there  are  two  types, 
a  slender  and  a  stout,  which  breed  true  to  these  characters  and 
correspond  to  slight  pathogenic  variations.     Noguchi  has  succeeded 


Fig.  8o. — ^The  Spirochaeta  refringens  is  the  larger  and  more  darkly  stained 
organism,  while  the  lightly  stained  and  more  delicate  parasite  is  the  Spirochaeta 
pallida  {Treponema  pallidum).  P^om  a  chancre  stained  with  Wright's  blood 
stain.     (Hirsch — by  Rosenberger.) 


in  cultivating  the  Tr.  pall,  in  pure  culture  by  using  the  juice  from 
human  or  monkey's  lesions  or  from  the  syphilitic  orchitis  of  rabbits. 
This  he  grows  in  serum  water  or  serum  agar,  to  which  has  been 
added  fresh  tissue  of  rabbit.     The  organism  grows  as  fine  fibrils  in 


RELAPSING   FEVER   ORGANISM  221 

arborescent  colonies.  These  can  be  selected  pure  by  cutting  the 
tube  and  the  agar  column.  Motion  is  of  screw  and  serpentine 
character.  No  odor  or  spores  are  produced.  This  organism  must 
be  imagined  and  remembered  as  a  corkscrew  and  not  a  waving  line. 
The  Gram  stain  is  negative. 

The  Spirochceta  refringens,  which  has  been  also  cultivated  by 
Noguchi  and  thought  by  him  to  be  a  Treponema  also,  grows  without 
fresh  animal  tissue  in  a  short  time  and  produces  no  odor. 

Pathogenesis. — It  has  been  found  in  chancre,  condylomata,  and 
mucous  patches  in  the  early  stages  of  syphilis;  also  in  the  blood, 
blister-fluids,  spleen,  bone  marrow,  liver,  thymus  gland,  and  lym- 
phatic glands.  Investigators  claim  that  it  exists  in  smegma  and 
other  foul  secretions,  but  this  has  not  been  confirmed.  Associated 
with  this  organism,  in  nearly  every  case,  is  a  coarser  looking  larger 
spirochaete  (Treponema),  which  stains  deeper,  and  has  been  called 
the  Spirochaeta  (Treponema)  refringens. 

In  a  series  of  experiments,  Metchnikoff  and  Roux  caused  abortion 
of  the  chancre  following  inoculation  of  syphilitic  virus  on  the  eyelid 
of  a  chimpanzee,  by  calomel  inunction  carried  out  less  than  one  hour 
after  the  infection;  a  solution  of  sublimate  has  not  the  same  pro- 
phylactic property. 

It  does  not  require  any  intermediate  host  for  transmission  as  do 
the  recognized  animal  parasites  of  malaria  and  filariasis,  etc. 

RELAPSING  FEVER  ORGANISM. 

European  Relapsing  Fever: — Caused  by  Spirochaeta  obermeieri, 
transmission  not  exactly  known. 

African  Relapsing  Fever: — Caused  by  Sp.  duttoni,  transmitted 
by  tick  Ornithodorus  moubata. 

American  Relapsing  Fever: — Caused  by  Sp.  Novii|  transmission 
not  known. 

Botnbay  Relapsing  Fever. — Caused  by  Sp.  carteri,  transmission 
not  known. 


222  ANIMAL   PARASITES 

Morphology. — These  are  probably  all  transmitted  by  ticks  or 
related  insects.  They  have  lately  been  cultivated  and  retain  some- 
what of  their  virulence  for  monkeys  and  rodents.  Close  studies 
have  placed  them  among  the  Spirochetes,  since  they  possess  an 
undulating  membrane,  some  divide  in  longitudinal  maimer  and 
since  an  insect  is  necessary  for  their  transmission.  They  are 
elongated,  flexible,  corkscrew-like,  serpentine  and  vibratory  in 
motility,  and  do  not  form  spores.  They  are  stained  with  reasonable 
ease  by  polychrome  methods  but  not  by  Gram's  method.     They 


Fig.    8i. — Spirilla   of   relapsing   fever   from   blood   of   a   man.     (KoUe   and 

Wassermann.) 


measure  from  io-40/i  in  length  and  about  ifi  in  breadth.     Coils 
vary  from  6-20.     The  American  type  is  smaller  than  the  rest. 

Transmission. — The  tick  which  transmits  these  organisms 
becomes  infective  in  one  week  after  biting  a  patient  and  remains 
so  all  its  life;  its  young  are  also  infective.  The  types  of  disease 
vary  but  little.  In  all  these  is  a  relapsing  fever  with  periods  of 
apyrexia  in  between.  During  the  fever  the  spirochaetes  are  swim- 
ming free  in  the  blood  and  disappear  in  the  afebrile  interval. 


MALARIAL    PARASITES  223 

Cultivation. — They  are  cultivated  in  the  manner  given  for  Trep. 
pallidum  by  Noguchi,  by  adding  citrated,  therefore  defibrinated, 
blood  to  serum  or  ascitic-fluid-fresh-tissue-agar.  They  breed  true 
to  type.  They  remain  alive  several  days  under  favorable  artificial 
conditions  but  cannot  be  cultivated  after  they  have  left  the  body  a 
few  hours  without  being  on  suitable  culture  media. 

The  periods  of  fever  last  from  five  to  seven  days,  when  a  crisis 
occurs.  After  an  apyrexial  period  the  fever  recurs.  The  spiro- 
chaetae  are  found  in  great  numbers  in  every  microscopical  field. 

In  the  apyrexial  period  the  spleen  becomes  engorged  and  the 
leucocytes  devour  the  parasites.  Monkeys  with  excised  spleens  are 
more  susceptible  to  infection  than  others. 

Immunity. — The  blood  from  rats  that  have  been  immunized  by 
repeated  injections  of  blood  from  spirochetal  rats,  if  injected  into 
other  rats,  is  capable  of  conferring  an  immunity  on  them  by  causing 
spirochaetes  to  disappear  from  their  blood. 

SPOROZOA. 

The  most  important  of  this  family  are  the  malarial  parasites 
(which  belong  to  the  order  Haemosporidia) ,  and  the  Coccidia. 

In  general  the  sporozoa  are  unicellular  organisms  that  lead  a 
parasitic  existence  in  the  tissues,  especially  cells,  of  higher  animals. 
They  ingest  liquid  food,  have  no  cilia  in  the  adult  stage,  and  flagella 
are  possessed  only  by  the  males.  There  may  be  one  or  more  nuclei. 
Propagation  is  effected  by  spores,  but  budding  and  division  do 
occur,  though  rarely.     Alternate  generation  takes  place  frequently. 

MALARIAL  PARASITES. 

Haemosporidia  of  Man. — The  most  important  disease  caused 
in  human  beings  by  the  haemosporidia  is  malaria,  or  ague,  and  ex- 
cepting the  deserts,  mountains,  and  arctic  regions,  this  disease  is 
very  widely  distributed. 


224  ANIMAL  PARASITES 

Three  different  parasites  producing  different  clinical  entities 
are  known.  According  to  the  time,  frequency,  and  order  of  the 
outbreak  of  chills  and  fever,  various  clinical  names  have  been  given 
to  the  manifestation  of  the  disease.  Mannaberg  has  arranged  the 
following  scheme  to  show  the  different  forms  of  outbreaks.  The 
numbers  apply  to  the  paroxysms.  Each  developmental  cycle  is 
numbered  alike: 

I  I  I  I  I  I  I.  Simple  quotidian  fever. 

I  o  I  o  I  o  I.  Simple  tertian  fever. 

looiooiooi.  Simple  quartan  fever. 

I2I2I2I2.  Double  tertian  fever.     {Two  infections.) 

123123123.  Triple  quartan  fever.     {Three  infections.) 

120120120.  Double  quartan  fever.     {Two  infections.) 

The  figures  refer  to  days  on  which  paroxysms  of  fever  occur.  The 
o  represents  the  afebrile  day. 


PLASMODIUM  MALARIA  {Laveran). 

This  is  the  quartan  parasite,  and  produces  in  man,  in  cases  of 
one  infection,  paroxysms  of  fever  every  fourth  day. 

It  appears  in  the  blood,  after  a  paroxysm,  as  a  small  non-pig- 
mented  body  on  the  bodies  of  the  red  blood  cells.  It  has  feeble 
amoeboid  motion;  slowly  penetrates  the  corpuscle,  and  specks  of 
melanin  appear  in  its  protoplasm.  Forty-eight  hours  after  the 
attack  the  parasite  measures  from  one-half  to  two-thirds  the  size  of 
the  red  cell.  Sixty  hours  after  the  paroxysm — twelve  before  the 
next — the  parasite  completely  fills  the  red  cell,  leaving  only  a  narrow 
rim,  which  later  on  disappears.  Six  hours  before  the  next  paroxysm, 
schizogony  begins.  The  grains  of  melanin  are  arranged  like  the 
spokes  of  a  wheel,  and  then,  leaving  the  radii,  crowd  about  the  cen- 
ter (the  rest  of  the  cell  being  pigmendess)  gradually  dividing  into 


PLASMODIUM   VI VAX  225 

nine  or  twelve  pear-shaped  bodies,  or  merozoites.  These  separate 
from  each  other  and  individually  attack  a  fresh  red  cell,  and  this 
attack  brings  about  another  paroxysm  of  fever  seventy-two  hours 
after  the  previous  one.  The  grains  of  pigment  are  taken  up  by  the 
leucocytes,  and  deposited  in  the  spleen  and  bone  marrow. 

The  nucleus  of  the  parasite  may  be  seen  if  suitably  stained.  The 
double  or  triple  quartan  is  explained  by  the  fact  that  there  are  two 
or  three  groups  of  organisms  that  undergo  sporogony  at  periods 
separated  from  each  by  twenty-four  hours. 

PLASMODIUM  VIVAX  (Grassi). 

The  cause  of  tertian  fever  occurring  in  the  spring.  It  differs 
from  the  Plasmodium  malarice  because  of  shorter  period  (forty- 
eight  hours)  consumed  in  schizogony  (or  sporulation) ,  the  much 
greater  activity  of  the  amoeboid  movement,  and  the  affected  corpus- 
cles becoming  enlarged;  also  by  the  fact  that  many  of  the  melanin- 
bearing  stages  are  visible.  The  schizogony  is  rarely  apparent 
in  the  circulating  blood,  but  in  the  spleen  these  stages  are  easily 
seen.  There  are  from  fifteen  to  twenty  merozoites  (segmented 
bodies  or  spores)  which  are  arranged  in  an  irregular  heap,  but  not 
radially  hke  wheel  spokes.  The  merozoites  are  smaller  than  the 
quartan  variety  and  are  more  numerous.  The  flagellated  form  can 
but  rarely  be  seen  in  the  freshly  drawn  blood.  If  some  blood,  con- 
taining the  large  extra-corpuscular  bodies,  is  put  in  a  moist  chamber, 
they  throw  out  flagella.  These  flagella  are  really  microgametes  and 
are  sexually  active.  The  extra-corpuscular  bodies  are  parUy  mac- 
rogameles,  and  if  they  become  flagellated  they  are  called  polymites, 
and  are  the  micro gametocytes.  The  merozoites  or  spores,  finally 
burst  forth  from  the  erythrocytes,  starting  again  another  cycle 
(attended  with  a  paroxysm  of  fever).  These  spores  appear  in  the 
freshly  invaded  corpuscles  as  hyaline  bodies  with  slight  movement. 
As  they  grow  in  size,  pigment  appears  in  the  protoplasm.  Certain 
of  these  do  not  break  up  into  merozoites,  or  spores,  but  become  extra- 
15 


226  ANIMAL   PARASITES 

cellular  bodies  and  polymites  if  they  develop  flagella  in  the  moist 
chamber.  There  may  be  two  infections  in  which  schizogony  occurs 
every  other  day  in  alternate  days  12121212. 

PLASMODIUM  FALCIPARUM. 

The  Plasmodium  of  aestivo-autumnal  fever,  or  pernicious  malarial 
fever,  also  called  tropical.  The  outbreaks  of  this  occur  irregularly. 
The  disease  produced  by  them  is  very  much  more  malignant  and  is 
harder  to  cure.  The  young  spore  appears  in  the  corpuscle  as  a 
small  hyaline  body,  smaller  than  the  other  forms  and  much  more 
active.  The  size  and  shape  of  the  red  cells  are  little  if  any  altered 
but  they  become  granular  and  polychromatophilic.  The  pigment 
is  very  finely  granular  and  the  body  frequently  presents  the  signet 
ring  appearance.  There  may  be  more  than  one  parasite  to  a  red 
cell.  The  cycle  of  development  (schizogony)  is  twenty-four  to 
forty-eight  hours.  The  plasmodium  in  its  schizogony  divides  into 
7-25  merozoites  or  spores,  and  are  arranged  in  a  spore-like  form. 
The  extra-corpuscular  bodies  may  resemble  a  crescent  or  sickle;  this 
form  is  very  characteristic  of  aestivo-autumnal  fever.  There  are 
two  forms  of  these  crescents,  one  delicate,  the  male  and  one  larger 
and  ovoid,  the  female.  They  are  very  resistant  to  quinine  and 
persist  for  a  long  period  in  the  blood.  Plasmodia  undergoing 
schizogony  are  often  found  in  the  brain  capillaries  after  death,  which 
accounts  for  the  cerebral  symptoms  in  such  cases.  This  form  can 
be  differentiated  from  the  others  by  the  irregular  and  pernicious 
type  of  fever  produced;  by  its  great  resistance  to  quinine;  the  fewer 
number  of  merozoites;  the  finely  granular  appearance  of  the  pig- 
ment; the  relatively  small  size  of  the  young  intra-corpuscular  body; 
and,  by  the  ring  shape  of  some  of  the  young  forms. 

Often,  in  blood  from  malarial  cases,  pigmented  leucocytes  are 
seen,  and  ghost,  or  shadow,  red  corpuscles  from  which  the  haemo- 
globin has  been  dissolved  are  often  met  with.  Spherical  extra-cor- 
puscular bodies  become  flagellated  (polymites)  in  freshly  drawn 


PLASMODIUM  FALCIPARUM  227 

blood.  The  parasite  may  be  studied  in  fresh  film  preparations, 
and  by  staining  dried  films  by  methylene  blue  and  eosin,  Romanow- 
sky's,  or  Jenner's  methods.  They  are  much  more  frequent  in  the 
pyrexial  period,  and  when  quinine  has  not  been  given. 

The  various  plasmodia  are  transmitted  to  man  invariably  by  the 
anopheles  mosquito,  in  the  bodies  of  M^hich  they  undergo  a  different 
(sexual)  existence.  It  has  been  positively  demonstrated  that  the 
various  plasmodia  undergo  an  alteration  of  generations  and  require 
two  different  hosts  for  their  development,  i.e.,  mosquito,  man. 

The  asexual  development,  or  schizogony,  takes  place  in  the  blood 
of  man,  the  sporogony,  or  sexual  development,  in  the  body  of  the 
anopheles  mosquitoes,  the  bite  of  which  sets  up  an  infection  in  man, 
since  the  sporozoites  of  the  various  plasmodia  are  developed  in  the 
salivary  glands  of  these  mosquitoes.  In  the  act  of  biting,  the  sporo- 
zoites reach  the  erythrocytes  where  they  become  the  intra-corpus- 
cular  hyaline  bodies  beginning  again  their  asexual  cycle  of  develop- 
ment  in  the  blood. 

That  the  mosquito  is  the  intermediate  host  of  the  malarial  para- 
site and  that  the  infection  in  man  follows  bites  by  infected  mosqui- 
toes has  been  abundantly  proven.  The  mosquitoes  that  act  in  this 
way  are  the  various  Anopheles;  the  Anopheles  maculipennis  being  the 
offender  most  frequently.  The  freshly  formed  schizonts  in  the 
blood  of  an  infected  man  are  conveyed  into  the  intestines  of  the 
mosquito.  Here  sexual  reproduction  of  the  parasite  begins.  The 
male  elements,  filamentous  micro  gametes  ^  penetrate  the  female  ele- 
ments, macro  gametes  (spheres),  and  after  a  time  become  mobile  fusi- 
form bodies,  ookinets.  These  bore  into  the  intestinal  walls  of  the 
mosquito  and  there  remain.  After  a  time  they  are  converted  into 
round  bodies,  or  oocysts.  The  nucleus  of  the  oocysts  divides  rapidly 
and  other  daughter  nuclei  are  formed,  and  new  cells  called  sporo- 
blasts.  After  about  eight  days  these  form  the  sporozoites.  The 
number  of  sporozoites  in  each  oocyst  varies  from  hundreds  to  many 
thousands  (often  10,000).  These  oocysts  burst  and  the  sporozoites 
in  the  circulation  find  their  way  to  the  salivary  glands  of  the  mos- 


DESCRIPTION  OF  FIG.  82. 

Life  history  of  malaria  parasite,  Plasmodium,  i,  Sporozoite,  introduced 
by  mosquito  into  human  blood,  the  sporozoite  becomes  a  schizont;  2,  young 
schizont;  3,  young  schizont  in  a  red  blood  corpuscle;  4,  full-grown  schizont; 
5,  nuclear  division;  6,  spores,  or  merozoites,  from  a  single  mother-cell;  7,  young 
macrogamete  (female),  from  a  merozoite,  and  situated  in  a  red  blood  cor- 
puscle; 7a,  young  microgametoblast  (male);  8,  full-grown  macrogamete;  8a, 
full-grown  microgametoblast;  9,  mature  macrogamete;  9a,  mature  micro- 
gametoblast; 96,  resting  cell,  bearing  six  flagellate  microgametes  (male); 
10,  fertilization  of  a  macrogamete  by  a  motile  microgamete;  the  macrogamete 
next  becomes  an  ookinete;  11,  ookinete,  or  wandering  cell,  which  penetrates 
into  the  wall  of  the  stomach  of  the  mosquito;  12,  ookinete  in  the  outer  region  of 
the  wall  of  the  stomach,  i.e.,  next  to  the  body  cavity;  13,  young  oocyst,  derived 
from  the  ookinete;  14,  oocyst,  containing  sporoblasts,  which  develop  into  sporo- 
zoites;  15,  older  oocyst;  16,  mature  oocysts,  containing  sporozoites;  17,  transverse 
section  of  salivary  gland  of  an  Anopheles  mosquito,  showing  sporozqites  of  the 
malaria  parasite  in  the  gland  cells  surrounding  the  central  canal. 

1-6  illustrate  schizogony  (asexual  production  of  spores);  7  16,  sporogony 
(sexual  production  of  spores) . 

(FoLSOM — After  Grassi  and  Leuckart,  by  permission  of  Dr.  Carl  Chun.) 


MALARIAL  PARASITE 


229 


Fig.  82. 


230 


ANIMAL   PARASITES 


quito.  When  a  mosquito  bites  a  human  being  they  are  introduced 
into  the  blood  where  they  are  quickly  transformed  into  the  intracellu- 
lar hyaline  bodies  and  begin  their  asexual  sporogony  in  the  blood. 
Each  developmental  cycle  causing  a  febrile  paroxysm  either  every 
day  or  alternate  days,  or  on  every  fourth  day,  etc.,  depending  on  the 


Fig.  83. — Coccidium  hominis,  fron  intestine  of  rabbit:  i,  a  degenerate  epi- 
thelial cell  containing  two  coccidia;  2,  free  coccidium  from  intestinal  contents; 
3,  coccidium  with  four  spores  and  residual  substances;  4,  an  isolated  spore;  5, 
spore  showing  the  two  falciform  bodies — X1140.  (From  Railliet,  in  Tyson's 
Practice.) 


character  of  the  organisms  and  the  number  of  infections.  To  pre- 
vent spread  of  malaria,  mosquitoes  must  be  prevented  from  reach- 
ing individuals  infected  with  malaria  and  those  not  infected.  Screens 
accomplish  this  best.  The  larva  of  the  mosquito  develops  in  stag- 
nant water.     To  prevent  the  development  of  these  young  mosqui- 


COCCIDIUM  231 

toes  oil  should  be  poured  on  the  water,  thus  cutting  off  the  air  and 
means  of  respiration. 

Boss,  of  New  Orleans,  claims  to  have  successfully  cultivated 
malarial  plasmodia  of  the  species,  vivax  and  falciparum  by  the  use 
of  human  blood.  He  has  also  succeeded  when  using  Locke's  fluid 
minus  calcium  chloride  plus  ascitic  fluid.  One-half  percent  dex- 
trose is  usually  added.  The  blood  is  drawn,  so  that  it  can  be 
defibrinated,  into  small  flat  bottom  tubes.  These  are  incubated  at 
40°  C.  The  column  of  fluid  is  1-2  inches  high,  the  clear  serum  layer 
being  1/2  inch  at  least.  The  parasites  grow  in  the  upper  layer  of 
the  cellular  sediment.  Undiluted  serum  and  leucocytes  are  lytic 
for  Plasmodia.  For  renewed  cultures  these  must  be  removed  but 
uninjured  red  cells  must  be  added.  Only  the  asexual  division  has 
been  observed.  Leucocytes  phagocyte  all  free  parasites  under 
artificial  conditions. 

COCCIDIUM. 

Coccidium  hominis  is  another  member  of  the  sporozoa  that  occa- 
sionally infects  man.  Coccidia  are  infectious  also  for  horses,  goats, 
oxen,  sheep,  pigs,  guinea  pigs,  weasels  and  rabbits.  The  organism 
is  essentially  a  cell  parasite  inhabiting  the  cells  of  the  gastro-intes- 
tinal  tract  by  preference,  chiefly  the  liver  and  intestinal  mucous  mem- 
branes. They  lead  a  sexual  and  asexual  existence  like  the  malarial 
parasites  (alternate  generation) .  The  young  sickle-shaped  nucleated 
sporozoite  penetrates  an  epithelial  cell,  where  it  gradually 
develops,  ultimately  dividing  into  numerous  sporozoites.  This  is  the 
asexual  stage  of  development  (schizogony),  the  sexual  stage  being 
called  sporogony. 

The  sporozoites  are  differentiated  into  the  two  sex  elements. 
These  are  large  granular  appearing  cells,  the  male  being  smaller, 
divides  into  numerous  flagellated  microgametes  that  penetrate  the 
female  granular  cells,  macrogametes,  and  fertilize  them.  These 
fertilized  macrogametes,   or  zygotes  form  capsules  and  become 


232 


ANIMAL   PARASITES 


oocysts  which  divide  into  numerous  sporoblasts,  changing  into  sic- 
kle-shaped sporozoites  upon  liberation. 

The  coccidia  are  easily  demonstrable  in  tissue  and  in  feces.  They 
produce  in  man  occasionally  a  fatal  disease  infecting  the  liver  and 
intestines.     Cattle  sometimes  die  from  haemorrhagic  dysentery  due 


Fig.  84. — Development  of  coccidium  cuniculi:  a,  b,  c,  young  coccidia  in  epi- 
thelial cells  of  gall  duct;  d,  e,f,  fully  grown  encysted  coccidia;  g,  h,  i  k,  I,  show- 
ing development  of  spores;  m,  isolated  spore,  greatly  magnified,  showing  the 
two  falciform  bodies  (pseudonavicella;  sporozoites)  in  natural  position;  n,  a  spore 
compressed  so  as  to  separate  the  two  sporozoites,  o,  a  sporozoite  or  falciform 
body  with  y,  its  nucleus.     (From  Railliet  after  Balbiani — in  Tyson's  Practice.) 

to  one  of  the  coccidia.     The  disease  is  transmitted  by  the  ingestion 
of  food  contaminated  by  feces  containing  the  sporozoites. 
Acid  fuchsin  stains  the  sporozoa. 


BABESIA  OR  PIROPLASMA  BIGEMINA. 

A  protozoon  supposed  to  be  the  cause  of  spotted  fever  in  the  valley 
of  the  Bitter  Root  river,  Montana.  This  cattle  disease  is  a  febrile 
one  characterized  by  an  irregular  fever  range,  by  muscular  pains, 


BABESIA   OR  PIROPLASMA  BIGEMINA  233 

arthritic  involvement,  petechia,  and  purpura  in  the  skin.  It  is 
supposedly  infectious,  but  not  contagious.  Its  cause  is  considered 
by  Wilson  and  Chowning  to  be  the  protozoon  Pyroplasma,  which 
occurs  in  the  blood  of  infected  individuals.  It  appears  within  the 
erythrocytes  and  they  resemble  hyaline  bodies  of  malaria.  They 
are  from  i[i  to  2//  in  length,  sometimes  from  four  to  sixteen  bodies 
are  found  within  a  single  cell.  They  grow  gradually  larger  and  then 
exhibit  amoeboid  motion  with  pseudopodia  formation. 

By  injecting  blood  from  an  infected  man  into  rabbits,  the  latter 
become  infected,  and  the  parasites  are  found  in  the  blood.  It  is 
believed  by  the  discoverer  that  the  parasites  are  conveyed  from  the 
gopher  Spermophilus  columbianus  to  man  by  the  means  of  ticks,  the 
Margaropus  annulatus. 


CHAPTER  X. 


THE  FILTERABLE  VIRUSES. 


This  general  term  means  that  the  virus  of  a  disease  can  pass 
through  a  porcelain  filter  and  usually  that  it  cannot  be  see.n  by  the 
microscope.  It,  however,  does  not  mean  that  it  is  invisible  at  all 
stages  since  in  one  case  at  least  we  have  been  able  by  means  of  the 
ultramicroscope  to  see  what  is  almost  certainly  the  particular  causal 
agent.  Again  it  is  said  the  spirochaetes  when  young  will  traverse 
porcelain  filters.  The  term  will  cover  in  this  chapter  those  diseases 
of  importance  to  man  whose  causal  agents  cannot  be  morphologic- 
ally described,  but  whose  characters  are  more  or  less  well  known. 
The  list  of  diseases  caused  by  sub  microscopic  agents  is  as  follows: 
African  horse  sickness,  swamp  fever  of  horses,  catarrhal  fever  of 
sheep,  yellow  fever.  Dengue,  three-day  fever,  typhus  fever,  polio- 
myelitis, rabies,  variola,  with  its  congeners  vaccinia  and  animal 
pox,  hog  cholera,  foot  and  mouth  disease,  fowl  plague,  fowl  diph- 
theria, transplantable  sarcoma  and  leukemia  of  fowls,  cattle  plague, 
trachoma,  pleuropneumonia  of  cattle,  molluscum  contagiosum, 
measles,  scarlet  fever,  guinea  pigs  epizootic  and  some  diseases  of 
plants.  As  said  above,  only  the  diseases  transmissible  to  human 
beings  are  reviewed. 

Hydrophobia. — This  disease  has  long  been  considered  to  be  an 
infectious  one,  but  the  causal  parasitic  agent  has  never  been  discov- 
ered. It  is  commonly  found  in  dogs,  cats,  wolves,  rabbits,  etc.,  but 
other  domestic  animals,  and  man  may  become  infected.  It  is  a 
disease  of  the  central  nervous  system,  highly  infectious,  always 
following  a  bite  or  other  injury  in  which  the  skin  is  broken,  and 
in  which  lesion  the  virus  may  be  deposited.  Infection  may  be 
caused  by  injecting  emulsified  infected  nerve  tissue  (brain)  into 

234 


HYDROPHOBIA  235 

susceptible  animals  (rabbits  or  monkeys).  The  disease  is  always 
fatal  after  it  is  well  established.  Well-marked  histological  lesions 
of  the  central  nerve  tissues,  particularly  the  large  ganglia,  have 
been  found  by  Van  Gehutchen  and  Nelis,  and  Ravenel  and  Mc- 
Carthy. If  emulsified  brain  tissue  from  an  animal  that  has  died  of 
hydrophobia  is  filtered  through  a  "germ-proof"  filter  the  filtrate  is 
capable  of  setting  up  the  disease  in  a  healthy  animal  if  it  is  injected 
into  it.  By  long  centrifugation  of  emulsified  infected  brain  tissue, 
the  supernatant  fluid  loses  its  power  of  reproducing  the  disease  on 
injection.  Virus  may  also  be  found  in  mammary  and  lacrymal 
secretions,  pancreas,  cerebro-spinal  fluid  and  aqueous  humor. 

The  organism  is  toxic  in  character,  since  filtrates  sometimes  fail 
to  produce  transmissible  disease,  but  emaciation,  paralysis,  and 
death  are  caused  by  their  injection  into  rabbits,  the  tissues  of  which, 
in  turn,  are  not  infectious. 

The  unknown  organisms  are  rather  resistant  to  agents  that  are 
germicidal.  They  are  destroyed  in  fifty  minutes  by  a  5  percent 
carbolic  solution,  and  in  three  hours  by  a  1-1,000  corrosive  sublimate 
solution.  Direct  sunlight  kills  them  quickly,  so  do  radium  emana- 
tions. The  latter  have  been  used  as  a  curative  measure  with 
reputed  success.  A  temperature  from  52^-58°  C.  for  one-half  hour 
destroys  them,  but  they  resist  extreme  cold  of  liquid  air  ( — 312°) 
for  many  weeks.  Pasteur  found  that  desiccation  attenuated  the 
virus.  Chlorine  kills  it  quickly,  while  glycerine  does  not.  The 
virus  may  be  increased  in  virulence  by  passing  the  "street  virus" 
of  dogs  through  a  series  of  rabbits.  Here  the  period  of  incubation 
decreases  from  three  weeks  to  six  days,  but  beyond  this  the  period 
does  not  become  less,  and  the  degree  of  virulence  from  the  virus  lead 
Pasteur  to  name  it  virus  fixe  (fixed  virus). 

Passing  the  virus  through  foxes,  cats,  and  wolves  also  intensifies 
the  virulence,  while  monkeys  and  chickens  attenuate  it. 

Negri  bodies,  protozoon  bodies  discovered  by  Negri,  are  found  in 
the  ganglionic  cells  of  rabid  animals.  These  bodies  stain  by  eosin, 
and  are  from  one  to  twenty-seven  microns  in  size,  being  generally 


236  THE   FILTERABLE   VIRUSES 

about  five  microns.  They  are  found  particularly  in  the  cornu  of 
Ammon;  in  Purkinje's  cells  in  the  cerebellum;  and  in  the  larger 
cells  of  the  cortex  of  the  cerebrum.  These  may  be  the  cause  of  the 
disease,  but  there  are  several  objections  to  this  hypothesis.  Their 
distribution  does  not  correspond  to  the  parts  of  the  nervous  system 
that  are  most  intensely  affected  in  hydrophobia,  i.e.,  medulla  and 
pons.  In  the  latter  locality  these  bodies  are  rarely  encountered. 
They  are  not  found  invariably  in  animals  dead  from  rabies,  and  are 
considered  to  be  too  large  to  pass  through  a  Berkefeld  filter;  this 
latter  view  may  not  be  a  correct  one.  The  finding  of  these  bodies 
has  been  considered  by  Negri  to  be  good  grounds  for  considering 
the  case  to  be  hydrophobia.  The  rapid  diagnosis  of  the  disease  in 
animals  can  only  be  effected  by  killing  them  and  examining  the 
nervous  tissues,  or  inoculating  other  animals  with  them.  Histo- 
logically, three  marked  changes  may  be  noted:  i.  The  finding  of 
the  Negri  bodies.  2.  The  finding  of  the  degeneration  of  the  cells 
of  the  larger  ganglia  with  the  proliferation  of  the  endothelial  cells 
lining  the  ganglionic  spaces  (Van  Gehutchen  and  Nelis).  3.  The 
finding  of  certain  tubercles  in  the  medulla,  which  are  called  Babes 
tubercles,  though  these  are  not  wholly  characteristic,  as  they  are 
found  in  other  diseases.  Hydrophobia  is  transmitted  from  the  site 
of  the  wound  to  the  central  nervous  tissues  by  the  nerves,  and  the 
incubation  period  varies  with  the  distance  of  the  wound  from  the 
central  nervous  system. 

Immunity  against  infection  and  the  development  of  the  disease 
after  the  reception  of  an  infected  wound,  may  be  accomplished  by 
Pasteur's  method.     (See  chapter  on  vaccine.) 

Yellow  Fever. 

That  this  disease  is  caused  by  a  parasite  there  can  be  no  doubt. 
It  is  highly  infectious  and  largely  confined  to  the  tropical  regions 
of  the  western  hemisphere  and  in  parts  of  Africa. 

Like  several  of  the  protozoon  parasites,  this  one  is  unquestionably 
spread  by  mosquitoes,  and  it  has  been  definitely  determined  by 


YELLOW  FEVER  237 

Carrol  and  Reed  that  the  female  Stegomyia  fasciata  (also  called 
Steg.  calopus)  is  the  means  of  its  propagation.  Carrol  believes  that 
the  undiscovered  parasite  of  yellow  fever  is  of  the  animal  kingdom, 
for  the  following  reasons:  i.  It  is  absolutely  necessary  for  its  con- 
tinued existence  that  it  undergoes  alternate  generation  in  man  and  in 
the  Stegomyia  mosquito.  This  is  peculiar  to  the  sporozoa.  2.  The 
fact  that  two  weeks  must  elapse  before  the  mosquito  is  capable  of 
infecting  man  is  evidence  that  a  cycle  of  development  of  the  unknown 
parasite  is  taking  place  in  the  mosquito.  3.  The  limitation  of  the 
cycle  of  development  of  the  parasites  to  a  single  genus  of  the  mos- 
quito and  to  a  single  vertebrate  (man)  conforms  to  a  natural  zoologic 
law,  and  this  does  not  conform  to  our  knowledge  of  the  life  history  of 
bacteria.  4.  The  effects  of  climate  and  temperature  on  the  life 
history  of  the  stegomyia,  and  on  the  rate  of  development  of  the 
parasites  in  the  bodies  of  the  mosquitoes  are  exactly  the  same  as  the 
effects  of  the  same  conditions  on  the  anopheles  mosquito  and  the 
malarial  parasite.  Without  the  stegomyia  there  can  be  no  yellow 
fever.  Infection  requires  the  fulfilling  of  the  following  conditions: 
I.  By  the  bite  of  the  mosquito  providing  the  insect  has  fed  on 
the  blood  of  a  yellow  fever  patient  within  the  first  three  days  of 
the  fever.  2.  The  disease  is  -not  transferred  immediately,  but  a 
definite  incubative  period  of  more  than  eleven  days  must  elapse 
before  the  mosquito  can  transfer  the  disease.  After  twelve  days 
the  mosquito  has  been  found  to  be  infected  for  at  least  fifty-seven 
days.  3.  Yellow  fever  cannot  be  carried  by  fomites.  4.  Yellow 
fever  may  be  produced  in  a  healthy  man  by  the  subcutaneous 
injection  of  blood  from  a  yellow  fever  case  (parasites  in  the  blood) . 

5.  The  serum  of  a  yellow  fever  patient  filtered  through  a  very 
fine  Berkefeld  or  porcelain  filter  is  still  capable  of  setting  up  the  dis- 
ease if  injected,  proving  that  the  infection  agent  is  submicroscopic. 

6.  An  attack  of  yellow  fever  produced  by  the  bite  of  a  mosquito 
confers  immunity  against  subsequent  infection.  7.  The  period 
of  infection  is  usually  three  days  but  may  be  from  two  to  six  days. 
8.  A  house  or  ship  may  be  said  to  be  infected  with  yellow  fever 


238  THE   FILTERABLE  VIRUSES 

only  when  there  are  present  stegomyia  capable  of  conveying  the 
parasite  of  the  disease.  9.  The  spread  of  yellow  fever  may  be 
prevented  by  destroying  the  stegomyia  and  preventing  egress  and 
ingress  of  the  insects  from  yellow  fever  patients  to  the  non-immune. 
10.  No  insect,  other  than  the  stegomyia,  has  been  found  to  be  con- 
cerned in  the  spread  of  yellow  fever. 

Yellow  fever  is  a  tropical  or  subtropical  disease,  because  the 
stegomyia  is  confined  to  these  regions,  and  the  disease  is  found  in  low 
moist  localities  rather  than  those  that  are  drier  and  higher,  from 
the  fact  that  the  mosquito  inhabits  the  former  and  not  the  latter. 
Yellow  fever  dies  out  after  the  first  sharp  frost,  because  the  stego- 
myia are  then  either  killed  or  undergo  hibernation.  Many  conclu- 
sive experiments  by  Reed  and  Carrol,  by  Guiteras,  and  by  the 
French  Commission  have  proved  that  the  stegom3da  is  beyond 
doubt  the  cause  of  the  spread  of  the  disease.  No  immunity,  other 
than  the  actively  acquired  one,  is  known. 

Small-pox  and  Vaccinia. — These  two  diseases  must  be  consid- 
ered to  be  but  two  clinical  activities  of  one  unknown  specific 
micro-organism.  i* 

Certain  protozoonoid  bodies  have  been  seen  by  numerous  observ- 
ers, notably  by  VanderLoeff,  L.  Peiffer,  and  Guarnieri.  The  latter 
gave  the  name  Cytoryctes  vacciniae  s.  variolae.  In  the  deep 
layers  of  the  epithelial  cells  of  the  pustules  of  vaccinia  and  small-pox, 
in  the  experimental  lesions  on  the  corneae  of  rabbits,  and  in  the  proto- 
plasm of  the  cells,  these  bodies  are  found.  They  are  about  the  size 
of  a  micrococcus  and  exhibit  amoeboid  movements  in  hanging  drop 
preparations.  They  are  perfectly  characteristic  of  the  lesion  pro- 
duced in  vaccinia  and  are  not  found  in  other  diseased  conditions. 
Although  championed  by  the  great  authority  Prowaczek,  their 
protozoal  nature  is  not  accepted  by  all  authorities. 

In  variola  many  different  changes  occur  in  the  appearances  of 
these  cytoryctes,  suggesting  developmental  cycles. 

In  variola  they  are  often  intra-nuclear,  while  in  vaccinia  they  are 
never  foimd  within  the  nuclei. 


SCARLET   FEVER  •         239 

The  cycle  of  development  is  suggestive  of  the  development  of 
many  of  the  protozoa.  Stages  of  development  exhibiting  fusiform 
amoeboid  shapes  can  be  seen,  and  pseudopodia  can  be  detected  in 
the  process  of  developmental  stages  suggestive  of  gametocytes;  the 
union  of  the  gametes  and  the  ultimate  formation  of  the  zygote  can 
also  be  discerned. 

After  the  tenth  day  these  bodies  cannot  be  very  v^^ell  discerned 
in  the  tissues. 

There  is  reason  to  think  that  the  parasites  circulate  in  the  blood 
in  variola.  The  contagion  in  variola  is  thought  to  be  by  inhalation. 
It  is  certain  that  the  disease  can  be  produced  by  inoculation  with 
virus  from  a  case  of  small-pox.  The  contagion  exists  in  the  scales, 
pus  cells,  and  excretions  of  patients  ill  with  small-pox. 

If  the  virus  of  small-pox  is  introduced  into  a  monkey  and  then 
into  a  cow  the  disease  produced  is  not  variola,  but  vaccinia  (Monk- 
man).  The  hypothetical  organism  above  described,  cytoryctes, 
becomes  attenuated  in  the  cow,  so  that  it  is  incapable  of  producing 
variola,  but  vaccinia. 

Rabbits,  horses,  and  sheep  are  susceptible  of  inoculation  with 
the  virus  of  vaccinia  (see  vaccination).  Virus  may  be  tested  by 
rubbing  over  the  shaven  bellies  of  rabbits,  setting  up  minute  vesicles 
and  finally  crusts.     (Calmette.) 

The  two  viruses,  that  of  variola  and  that  of  vaccinia,  are  now 
thought  to  be  identical.  In  a  diluted  condition  it  is  filterable.  It 
resists  drying  for  weeks  and  glycerine  8-10  months.  It  is  de- 
stroyed at  57°  C.  in  15  minutes  and  easily  by  most  disinfectants. 
It  has  not  been  cultivated.  Passive  immunization  has  not  been 
achieved. 

Scarlet  Fever. 

Mallory  in  1903,  found  certain  bodies  in  the  skin  of  scarlet  fever 
cases.  These  bodies,  he  assumed,  were  protozoan  in  character 
and  were  the  etiological  cause  of  the  disease.  He  named  them 
Cyclasterion    Scarlatinale.     They    have    been    found    rather 


24©  THE   FILTERABLE    VIRUSES 

constantly  in  the  skin  of  scarlet  fever  cases,  also  in  the  skin  in  cases 
of  measles  and  in  anti-toxin  rashes. 

By  several  observers  they  have  been  considered  to  be  artefacts  or 
degeneration  products  in  the  epithelial  cells. 

The  virus  of  scarlatina  is  now  considered  to  be  filterable  and 
transmissible  to  monkeys. 

Dengue  Fever. — This  is  an  acute  infectious  disease  of  the 
tropics,  characterized  by  fever,  skin  eruptions,  rheumatoid  pains,  an 
afebrile  remission  and  a  febrile  end,  due  to  a  filterable  virus,  trans- 
mitted by  the  mosquito,  Culex  fagitans.  The  virus  is  in  the  blood 
stream.     One  attack  gives  immunity;  little  is  known  of  the  virus. 

Three-day  or  Sand-fly  Fever. — ^A  mild  infectious  disease 
chiefly  of  southeastern  Europe,  due  to  a  virus  which  will  pass 
through  a  bacteria-proof  filter  and  is  transmitted  by  the  sand- fly, 
Phlebotomus  pappatacii.     Cultures  have  not  been  obtained. 

Typhus  Fever  or  Spotted  Fever. — An  acute  epidemic  disease 
with  prolonged  course,  prostration,  a  macular  eruption,  ending  by 
crisis,  transmitted  by  the  body  louse,  Pediculus  vestamenti.  The 
virus  is  filterable  but  is  obtained  with  diflftculty.  It  is  found  best 
toward  the  end  of  the  fever.  It  may  be  transmitted  to  monkeys. 
It  has  not  been  cultivated.  It  is  destroyed  quickly  at  52°  C. 
Brill's  disease  is  a  mild  typhus  fever. 

Poliomyelitis. — An  acute  infectious  disease,  chiefly  of  children 
characterized  by  a  short  febrile  attack,  followed  by  a  rapidly 
appearing  paralysis  in  various  muscles.  Means  of  transmission 
from  child  to  child  is  unknown,  but  it  has  lately  been  shown  that 
the  stable  fly,  Stomoxys  calcitrans,  can  transmit  it  from  monkey  to 
monkey.  The  virus  is  in  the  central  nervous  system,  lymphatic 
system,  blood,  succus  entericus,  nasal  mucous  and  various  organs. 
It  is  said  to  be  constantly  in  the  nasal  mucosa  of  not  only  patients 
but  of  the  well  in  their  vicinity.  This  is  supposed  to  be  its  portal 
of  entry  to  the  body.  It  is  transmitted  to  monkeys  by  injecting 
emulsions  of  the  virus-containing  parts  into  the  brain,  blood- 
stream or  peritoneum.     It  can  be  filtered  through  porcelain.     It 


MEASLES  241 

has  not  been  cultivated.  It  resists  glycerine,  drying  and  autolysis. 
It  is  destroyed  at  50°  C.  in  one-half  hour.  Hexner  and  Noguchi 
have  succeeded  in  staining  a  very  minute  spirochaete — the  tissues 
of  monkeys  affected  with  this  disease. 

Active  artificial  immunity  and  some  passive  immunity  have  been 
obtained  but  these  are  not  of  therapeutic  value. 

Foot  and  Mouth  Disease. — ^An  acute  infectious  disease  of 
cattle,  characterized  by  a  vesicular  eruption  in  the  mouth  and 
around  the  crown  of  the  hoof.  It  may  be  transmitted  to  man  by 
the  use  of  milk  from  infected  cows.  It  is  also  directly  communica- 
ble. It  has  not  been  cultivated.  It  is  filterable ;  it  is  said  to  be  due 
to  the  Cytorrycetes.  It  is  destroyed  at  50°  C.  in  10  minutes,  easily 
by  freezing  and  ordinary  disinfectants.  One  attack  gives  immunity 
and  the  blood  is  said  to  contain  anti-bodies  which  will  be  protective 
to  other  animals. 

Trachoma.— An  infectious  inflammation  of  the  conjunctiva 
with  the  production  of  minute  but  visible  nodules  on  the  under 
sides  of  the  lids.  By  some  it  is  said  to  be  due  to  a  form  of  the 
influenza  bacillus,  by  others  to  an  invisible  virus.  It  is  directly 
communicable,  filterable  and  transmissible  to  monkeys.  It  has 
not  been  cultivated. 

Measles. — An  acute  eruptive  fever  due  to  a  filterable  virus 
which  is  found  in  the  blood,  buccal  and  nasal  secretions.  It  is 
transmissible  to  monkeys  by  inoculations  of  patient's  blood,  even 
before  the  Koplik  spots  appear.  It  persists  in  the  blood  until  after 
the  appearance  of  the  eruption.  It  resists  drying  and  freezing. 
It  is  destroyed  at  55°  C.  in  15  minutes;  it  has  not  been  cultivated. 
Immunity  follows  an  attack  but  no  passive  immunity  has  been 
reported. 

It  must  be  said  of  both  the  hypothetical  organisms  of  variola  and 
scarlatina,  that  if  they  are  the  cause  of  these  two  diseases  they  differ 
from  all  other  known  protozoan  parasites,  because  the  latter  require 
an  intermediate  host  for  the  transmission  of  the  parasite  from 
individual  to  individual  while  these  certainly  do  not. 
16 


j-BS-B-j^Sng  m 
SBQ  JO  uoipnpojj 

l+l+l+l+l      1+    +     II      1+ 

Indol 
Reaction 

Slight. 

Pronounced. 
Very  slight. 

Slight. 

Very 
pronounced. 
Faint  with- 
out nitrite. 

uopBuiioj-gjods 

1   1    i    1    II    1   1   1       II       1       1  +     ++  ! 

3 

1      u 

1 

Reaction 

Acid. 
Amphoteric. 

Acid. 

Acid. 
+  Acid. 

Acid. 

Acid. 

Amphoteric, 

later  alkal. 

Faintly  alkal. 

Faintly  acid. 

Faintly 

alkaline. 

Amphoteric. 

Faintly 
alkaline. 
Alkaline. 
+  Acid. 

uop 
-B|nSB03 

1    1  +4-  1  +  +  +       1  +       1      0+      +  1 

J5 

5 

u 
1 
1 

Cloudiness 

Moderate. 

Slight. 
Moderate. 

Marked. 

Moderate. 

Marked. 

Very  slight. 
Moderate. 

Moderate. 

Slight. 

Very 

marked. 

Moderate. 

+ 

sPITPd 

1    1    1    1    1    1  +  1    1        II        1       <1  1       +      . 

uijBpo 
JO  uoip^jgnbi^ 

1111+1+1+       1+      +       1+      ++ 

Aerobic 

and 

Anaerobic 

Growth 

OTqojg-Buv 

1  +  +  +  +  1  +  1  O     0+      +      ++     ++    ' 

Diqoi9V 

+++++++++     ++     +     ++     +1 

UIBIS  S.UIBO 

1 1 1 1 1 1 + 1 +     ++     +     ++     +1 

Flagella 



Many. 
A  few. 
Many. 

A  few. 
+  8 
One. 

Many. 
Many. 

Many. 

+ 

i 

a 

d 

Bact.  influenzae. 
Bact.  pneumoniae. 
Bact.  typhosus. 
Bact.  coli. 
Bact.  prodigiosum. 
Bact.  dysenterias. 
Bact.  violaceum. 
Bact.  enteritidis. 
Bact.  pyocyaneum. 

Bact.  Zopfii. 
Bact.  vulgare. 

Bact.  vulgare  /?  mirabilis. 

Bact.  erysipelatos  suum. 
Bac.  anthracis. 

Bac.  mycoides. 
Bac.  Botulinus. 

jB3B-JBSns  m 
SBO  P  uopDnpoijj 

+   \         III   +  +  +   1               1     II     1     1          1     1     1     1 

Indol 
Reaction 

"S^         .              .         . 

<i^  4J  ^J  *J   y  "5   aj  -t-I   ""  -M*  *^  ^J        ""              •" 

1     1  1  |§§|§|-5§S.'IM  s      s 

uopBuuoj-9Jods 

++     ++++++ 1           1       III       1 1 1 1 

1 

3 

1 

Acid. 
Faintly 
alkaline. 
Faintly  alkal. 
Strongly  alk. 
Faintly  alkal. 
Amphoteric. 
Faintly  acid. 
Amphoteric. 

Acid. 

Acid. 
Faintly  alkal. 

Acid. 
Amphoteric. 
Faintly  acid. 

Amphoteric. 
Faintly  alkal. 

uop 
-Bin3B03 

+  ++<+l  +  l+         +l<i<l|        II       + 

u 

1 

o 
U 

+ 
Very 
marked. 
Moderate. 
Moderate. 
Moderate. 
Moderate. 
Moderate    . 
Moderate. 
Moderate. 

Moderate. 
Slight. 

Moderate. 
Very  slight. 
Almost  clear. 

Clear. 

gpHFJ 

++     ++ 1 1 1       +              1 1 1 1        1 +       1 

m^Bpo 
JO  uopDBpnbiT 

1  +     +  +  +  +  +  +  +         +  1    1    1    1        111  + 

Aerobic 

and 

Anaerobic 

Growth 

3iqOJ9BUV 

+  +     +  +  +  +  +  +  +         +  +  +  +  +     +<]|0 

Diqoj9v 

1+     +++000+         +<+++     ++++ 

UTB^S  S^UIBJO 

+  -f     +  +  +  +  +  1    1            1  +  1+4-     +X  +  + 

J3 

1 

pj         ^ 

.      1  J         g^g^Sil-S^lg 

B 

d 

Bac.  cap.  aerogenes. 
Bac.  subtilis. 

Bac.  megatherium. 

Bac.  vulgatus. 

Bac.  mesentericus. 

Bac.  tetani. 

Bac.  Chauvoei. 

Bac.  oedematis  maligni. 

Vibrio  cholerae. 

Vibrio  proteus. 
Spir.  rubrum. 
Corynebact.  mallei. 
Corynebact.   diphtheriae. 
Cory  neb.  pseudodiph- 

theritic. 
Corynebact.  xerosis. 
Mycobact.  tuberculosis. 
Mycobact.  leprae. 
Actinomyces  bovis. 

DESCRIPTION  OF  PLATE  I. 


Malarial  Parasites. 


1.  Two  tertian  parasites  about  thirty-six  hours  old,  attacked  blood  corpuscles 
magnified. 

2.  Tertian  parasite  about  thirty-six  hours  old;  stained  by  Romanowsky's 
method.  The  black  granule  in  the  parasite  is  not  pigment  but  chromatin. 
Next  to  it  and  to  the  left  is  a  large  lymphocyte,  and  under  it  the  black  spot 
is  a  blood  plate. 

3.  Tertian  parasite,  division  form  nearby  is  a  polynuclear  leucocyte. 

4.  Quartan  parasite,  ribbon  form. 

5.  Quartan  parasite,  undergoing  division. 

6.  Tropical  fever  parasite,  (^stivo-autumnal.)  In  one  blood  corpuscle 
may  be  seen  a  smaller,  medium,  and  large  tropical  fever-ring  parasite. 

7.  Tropical  fever  parasite.  Gametes  half  moon  spherical  form.  Smear 
from  bone  marrow. 

8.  Tropical  fever  parasite  which  is  preparing  for  division  heaped  up  in  the 
blood  capillaries  of  the  brain. 

Asexual  Forms. 

9.  Smaller  tertain  ring  about  twelve  hours  old. 

10.  Tertian  parasite  about  thirty-six  hours  old,  so  called  amoeboid  form, 

11.  Tertian  parasite  still  showing  ring  fever,  forty-two  hours  old. 

12.  Tertian  parasite,  two  hours  before  febrile  attack.  The  pigment  is  begin- 
ning to  arrange  itself  in  streaks  or  lines. 

13.  Tertian  parasite  further  advanced  in  division.  Pigment  collected  in 
large  quantities. 

14.  Further  advanced  in  the  division.     (Tertian  parasite.) 


PLATE  I. 


DESCRIPTION  OF  PLATE  II. 


Malarial  Parasites. 


15.  Complete  division  of  the  parasite.     Typical  mulberry  form. 

16.  To  the  left  is  the  completed  division  form,  an  almost  developed  gamete, 
which  is  to  be  recognized  by  its  dispersed  pigment. 

17.  A  tertian  ring  parasite,  small  size  broken  up. 

18.  Three-fold  infection  with  tertian  parasite.  The  oval  black  granules 
are  the  chromatin  granules. 

19.  To  the  left,  tertian  parasite  with  large,  sharply  demarked,  and  deeply 
colored  chromatin  granules.  To  the  right,  tertian  parasite.  Both  thirty- 
six  hours  old.     Both  probably  gametes. 

20.  Tertian  parasite  thirty-six  hours  old,  ring  form. 

21.  Tertian  parasite  with  beginning  chromatin  division,  with  eight  chromatin 
segments. 

22.  Tertian  parasite  chromatin  division  farther  advanced  with  twelve  chrom- 
atin granules,  in  part  triangular  in  form. 

23.  Completed  division  figure  of  a  tertian  parasite.  Twenty-two  chromatin 
granules. 

24.  The  young  tertian  parasites  separating  themselves  from  each  other. 
The  pigment  remains  behind  in  the  middle. 

25.  Quartan  ring  parasite,  which  is  hard  to  differentiate  from  large  tropical 
ring  or  small  tertian  ring. 

26.  Quartan  ring  lengthening  itself. 

27.  Small  quartan  ribbon  form. 

28.  The  quartan  ribbon  increases  in  width.  The  dark  places  consist  almost 
entirely  of  pigment. 


PLATE  II. 


DESCRIPTION  OF  PLATE  III. 


Malarial  Parasite 


29,  30,  31.  The  quartan  ribbon  increases  in  width.  The  dark  places  consist 
almost  entirely  of  pigment. 

32.  Beginning  division  of  the  quartan  parasite  and  the  black  spot  in  the  middle 
is  the  collected  pigment. 

$;^.  Quartan  ring. 

34.  Double  infection  with  quartan  parasites. 

35.  Wide  quartan  band.  The  fine  black  stippling  in  the  upper  half  of  the 
parasite  is  pigment. 

36.  Beginning  division  of  the  quartan  parasite.  The  chromatin  (black  fleck) 
is  split  into  four  parts. 

37.  Division  advanced,  quartan  parasites. 

38.  Typical  division  figure  of  the  quartan  parasite. 

39.  finished  division  of  the  quartan  parasite.  Ten  young  parasites,  pigment 
in  the  middle. 

40.  Young  parasites  separated  from  one  another. 

41.  Small  and  med'um  tropical  ring,  the  latter  in  a  transition  stage  to  a  large 
tropical  ring. 

42.  Small,  medium  and  large  tropical  ring,  together  in  one  corpuscle. 


PLATE  III. 


DESCRIPTION  OF    PLATE  IV. 


Malarial  Parasite. 


43.  To  the  left  a  young  (spore)  tropical  parasite.  To  the  right  a  medium 
and  large  tropical  parasite. 

44.  An  almost  fully  developed  tropical  parasite.  The  black  granules  are 
pigment  heaps. 

45.  Young  parasites  separated  from  one  another.  Broken  up  division  forms 
twenty-one  new  parasites. 

46.  To  the  left  a  red  blood  corpuscle  with  basophilic,  karyochromatophilic 
granules.  Prototype  of  malarial  parasite.  On  the  right  a  red  blood  corpuscle 
with  remains  of  nucleus. 


Sexual  Forms  or  Gametes. 

47.  An  earlier  quartan  gamete  (microgametocyte  in  sphere  form),  female. 

48.  An  earlier  quartan  gamete  (microgametocyte),  male. 

49.  Tertian  gamete,  male  form  (microgametocyte). 

50.  Tertian  gamete,  female  (microgamete) . 

51.  Tertian  gamete  (microgametocyte)  still  within  a  red  blood  corpuscle. 

52.  Microgamete  tertian  within  a  red  blood  corpuscle. 

53.  Tropical  fever,  (^stivo-autumnal)  gamete,  half  moon  (crescent)  still 
lying  in  a  red  blood  corpuscle.  In  the  middle  is  the  pigment.  The  concave 
side  of  the  crescent  is  spanned  by  the  border  of  the  red  blood  corpuscle. 

54.  Gamete,  tropical  fever  parasite. 

55.  Gamete  of  tropical  fever  parasite  heavily  pigmented. 

56.  Gamete  of  the  tropical  fever  parasite  (flagellated  form),  microgametocyte 
sending  out  microgametes  (flagella  or  spermatozoon) . 


PLATE  IV. 


CHAPTER  XI. 

BACTERIOLOGY  OF  WATER,  AIR,  AND  SOIL. 

Bacteriological  examination  of  water  is  of  importance  for  the 
determination  of  the  presence  of  pathogenic  bacteria,  and  for  the 
enumeration  of  the  total  number  of  all  bacteria  contained  therein, 
the  latter  being  considered  an  index  of  the  purity  of  the  water. 

Several  well  known  pathogenic  bacteria  have  been  found  in  water; 
among  these  are  the  typhoid,  anthrax,  cholera,  plague,  and  colon 
bacilli,  also  the  pus  cocci.  Since  the  tetanus  bacillus  is  a  normal 
inhabitant  of  the  cultivated  soil  and  manure,  it  is  not  at  all  uncom- 
mon to  find  it,  at  times,  in  muddy  waters. 

Bacteriological  examinations  of  water  are,  in  a  measure,  very 
disappointing,  because  it  is  very  diflficult,  and  at  times  impossible 
to  determine  the  presence  of  the  typhoid  bacillus,  even  when  it  is 
certain  that  it  is  present,  having  been  added  to  water  to  be  ex- 
amined it  is  even  then  difficult  to  isolate. 

The  fact  that  the  colon  bacillus  is  always  found  in  water  con- 
taminated by  feces  is  a  great  help  in  the  recognition  of  polluted  water. 
In  the  case  of  typhoid  contamination  the  typhoid  bacillus  may  elude 
detection,  but  the  colon  bacillus  is  easily  found;  we  may  then  assume 
that,  since  it  is  impossible  for  typhoid  bacilli  to  reach  water  without 
the  colon  bacilli  that  water  having  no  colon  bacilli  is  also  free  from 
typhoid  bacilli.  Also  water  having  colon  bacilli  in  great  numbers 
is  contaminated  with  feces,  and  perhaps  typhoid  feces.  The  detec- 
tion of  the  colon  bacillus  is  therefore  of  prime  importance  in  the  ex- 
amination of  drinking  water.  Its  detection  is  simple.  Water  must 
be  collected  in  sterile  bottles,  using  every  precaution  against  acci- 
dental contamination.     Fermentation  tubes  are  employed,  contain- 

261 


.262  BACTERIOLOGICAL  EXAMINATIONS 

ing  bouillon  with  i  percent  of  glucose.  Into  a  series  of  these  tubes, 
varying  amounts  of  water  are  run  by  means  of  a  sterile  pipette,  2  c.c, 
I  c.c,  .5  c.c,  .1  c.c,  .01  cc,  of  water  being  used.  After  a  stay  of 
twenty-four  hours  in  the  incubator,  if  gas  appears,  the  bouillon 
should  be  examined  by  plate  cultures  for  the  colon  bacillus.  Lactose 
litmus  agar  is  used,  and  where  colonies  appear  that  redden  the 
litmus  and  resemble  the  colon  colonies  in  appearance,  they  are 
planted  in  milk,  fermentation  tubes,  peptone  solution,  neutral  red 
agar,  nitrate  solution,  and  gelatine,  and  the  various  reactions  in  the 
various  media  noted.  Some  idea  of  the  numerical  presence  of 
colon  bacilli  can  also  be  obtained.  Definite  quantities  of  the  raw 
water,  similar  to  those  used  in  the  fermentation  tubes,  may  be 
plated  directly  without  previous  incubation.  A  deeply  tinted  litmus 
lactose  agar  is  used  and  upon  this  medium  colon  bacillus  colonies 
appear,  small,  pink,  round  or  whetstone  shaped  surrounded 
by  a  pink  zone  or  halo.  Such  pink  colonies  are  fished  out  into 
the  different  media  as  above.  If  there  were  twenty  pink 
colonies  of  the  colon  type  upon  a  plate  of  litmus  lactose  agar 
that  had  been  seeded  with  i  c.c.  of  water  and  of  these  eight 
were  fished  and  determined,  with  the  discovery  that  four  only 
were  true  B.  coli,  we  would  assume  that  in  i  cc.  of  raw  water 
half  the  pink  growing  colonies  were  those  of  B.  coli  and  that  the 
water  contained  10  B.  coli  per  cubic  centimeter. 

The  significance  of  the  colon  bacilli  is  often  overestimated.  They 
are  found  in  all  rivers,  and  often  reach  streams,  wells,  and  even 
springs  by  contamination  from  the  barnyard,  or  manured  fields. 
Attempts  to  separate  colon  bacilli  from  human  and  animal  sources 
have  been  unsuccessful.  Some  authorities  use  streptococci  of  the 
fecal  type  as  pollution  indicators.     This  is  not  absolutely  reliable. 

Typhoid  bacilli  have  been  found  in  water.  One  way  that  is 
sometimes  successful  is  to  take  25  cc  of  a  4  percent  peptone  solution 
and  add  this  to  a  litre  of  the  water  to  be  examined;  from  this,  after 
twenty-four  hours  in  an  incubator,  plates  may  be  prepared  with  the 
agar  media  of  Drigalski  and  Conradi.     This  media  is  made  of  3 


TO   COUNT   BACTERIA   IN    WATER  263 

percent  agar,  to  which  has  been  added  nutrose  and  crystal  violet. 
In  the  following  order  add  two  litres  of  water  to  three  pounds  of 
beef,  straining  and  boiling  for  an  hour;  after  filtering,  add  twenty 
grams  each  of  nutrose  and  peptone,  and  ten  grams  of  salt.  Sixty 
grams  of  agar  are  then  added  and  the  mixture  boiled  and  filtered 
after  being  rendered  alkaline.  Boil  300  c.c.  litmus  solution  with 
thirty  grams  of  lactose,  mix  with  the  foregoing  and  alkalinize  with  a 
soda  solution,  and  then  add  to  this  4  c.c.  of  a  10  percent  soda 
solution,  and  20  c.c.  1-1,000  crystal  violet  (Hochst  B.).  Mix  these 
solutions  together,  tube,  and  pour  on  plates,  spreading  the  feces 
or  water  over  the  agar,  dry  and  invert  in  the  incubator  twelve  to 
twenty-four  hours. 

The  typhoid  colonies  in  this  medium  appear  less  granular  and 
dark  than  do  the  colon  colonies. 

Typhoid  colonies  1-3  mm.  in  size  appear  blue,  colon  colonies 
red,  all  other  bacteria  are  temporarily  inhibited  by  the  crystal  violet. 
Transfer  the  colonies  to  bouillon  and  test  wdth  a  highly  diluted 
serum  from  a  rabbit  artificially  immunized,  by  the  agglutination  test. 

To  Count  Bacteria  in  Water. 

The  sample  must  be  collected  in  a  sterile  bottle,  and  the  plates 
poured  immediately,  since  bacteria  multiply  enormously  after  a  few 
hours. 

Take  ^q-  c.c.  or  h  c.c.  or  i  c.c.  of  the  water  in  sterile  pipettes  and 
mix  with  a  tube  of  melted  gelatine  or  agar,  pour  quickly  into  cool 
sterile  petri  dishes  and  place  in  a  cool  dry  place.  The  American 
Public  Health  Association  also  recommends  the  use  of  +  i  percent 
agar  plates  grown  both  at  room  and  body  temperature.  The  counts 
for  the  two  are  averaged.  After  forty-eight  hours  count  the  colonies 
and  the  result  (after  multiplication  where  -^^  or  J  c.c.  of  water  was 
used)  will  be  the  number  of  bacteria  per  cubic  centimeter.  It  may 
be  necessary  to  dilute  the  water  5  or  10  times  before  pouring  plates. 
A  glass  plate  ruled  into  squares,  known  as  a  Wolffhiigel  plate,  should 
be  used  for  counting.     The  number  of  bacteria  in  potable  waters 


264  BACTERIOLOGICAL  EXAMINATIONS 

varies  in  many  ways,  according  to  the  amount  of  pollution,  or  albu- 
minous matter  in  the  water,  while  depth,  and  the  swiftness  with 
which  it  flows  are  conditions  that  modify  bacterial  contents.  The 
water  in  a  reservoir  becomes  almost  free  from  bacteria  during  the 
first  ten  days.  The  number  of  bacteria  diminishes  10  percent  per 
day  for  the  first  five  or  eight  days,  due  no  doubt  to  gravitation 
of  the  bacteria  to  the  bottom,  also  in  part  to  the  action  of  light, 
which  plays  an  important  role  in  the  destruction  of  the  bacteria 
of  water  supplies. 

In  general,  water  containing  less  than  100  bacteria  per  i  c.c. 
is  considered  to  be  from  a  deep  source,  and  uncontaminated  by 
drainage.  Deep  artesian  wells  often  contain  but  from  5  to  15 
bacteria  per  cubic  centimeter,  water  from  rivers  often  contain 
12,000  or  20,000,  depending  somewhat  upon  the  season  of  the 
year.  Rains  cause  an  augmentation  of  the  bacterial  content. 
Summer  causes  a  diminution. 

In  identifying  a  certain  water  supply  as  the  cause  of  an  epidemic 
of  typhoid,  the  number  of  bacteria  is  of  great  value  in  locating  the 
place  of  infection. 

The  efficiency  of  filters  in  large  municipal  water  supplies  is  only 
known  by  the  bacterial  content  of  the  effluent.  In  good  sand  and 
mechanical  (alum)  filters,  the  reduction  in  the  number  of  bacteria 
is  often  over  95  percent  (sometimes  99  percent).  Plate  cultures 
should  be  made  daily  from  every  filter  in  order  to  determine  how 
each  filter  is  performing.  Sand  filters  should  not  filter  more  than 
1,000,000  gallons  per  acre  a  day.  They  should  be  at  least  one 
metre  thick;  the  upper  half  inch  of  the  sand  performs  over  90  percent 
of  the  filtration,  due  to  a  certain  zooglea,  or  growth  of  bacteria. 
Cracks,  or  imperfections  in  the  filtei  beds  are  quickly  detected  by 
the  rapid  increase  of  the  number  of  the  bacteria  in  the  effluent. 
It  is  supposed  that  not  only  are  bacteria  filtered  by  the  sand  but 
that  destructive  changes  occur  in  the  filter  which  greatly  diminish 
the  number  of  bacteria.  A  filter  must  be  used  for  a  few  days 
before  it  becomes  efficient  or  "ripe."     After  a  time  it  becomes 


DISPOSAL   OF  SEWAGE  265 

inefficient  and  it  must  then  be  scraped,  finally  the  sand  must  be 
removed  and  washed. 

A  sand  filter  is  a  highly  efficient  means  of  water  purification.  It 
often  converts  a  foul  dirty  water  into  a  bright,  clean,  wholesome 
water  of  low  bacterial  content. 

Mechanical  filters  depend  for  their  efficiency  upon  the  addition 
of  aluminum  sulphate  to  the  water.  This  is  decomposed  by  the 
carbonates  and  aluminum  hydroxide  is  produced,  which  is  a  white 
jelly-like  flocculent  precipitate,  which  mechanically  entangles  bac- 
teria and  removes  them  from  the  water.  Mechanical  filters,  as  a 
rule,  are  highly  efficient.  Domestic  filters,  even  the  Pasteur,  are 
often  unreliable. 

In  time  of  epidemics  of  cholera  and  typhoid  even  filtered  water 
should  be  boiled  before  use,  as  it  was  found  by  experiments  in  the 
Medico- Chirurgical  Laboratories  that  typhoid  bacilli  live  longer  in 
filtered  water  than  in  bouillon;  they  may  even  live  three  months. 
The  fewer  the  number  of  other  bacteria  the  longer  will  typhoid  live. 
They  can  live  many  days  in  ordinary  river  water. 

Ice  may  contain  great  numbers  of  bacteria;  it  is  well  known  that 
freezing  does  not  destroy  pathogenic  bacteria,  such  as  the  typhoid 
bacillus.  Prudden  found  typhoid  bacilli  in  ice  after  100  days, 
although  the  number  was  greatly  reduced  over  that  placed  in  the 
ice  originally.  Many  are  squeezed  out  by  contraction  of  the  water. 
The  greatest  danger  from  ice  is  in  dirty  handling. 

Disposal  of  Sewage,  is  a  bacteriological  process  in  many  cases; 
either  the  sewage  may  be  treated  in  sand  filters  or  it  may  be  run  out 
on  land  where  over  200,000  gallons  may  be  disposed  of  on  an  acre 
of  land  a  day.  As  far  as  possible  nature  should  be  imitated  in  every 
way  and  the  breaking  up  of  masses  of  matter  in  sewage  may  be 
accomplished  in  the  septic  tank  process  in  which  active  oxidization 
of  the  matter  is  accomplished  by  bacteria.  It  appears  from  the 
observations  of  many  sanitarians  that  both  aerobic  and  anaerobic 
bacteria  are  necessary  to  finally  reduce  sewage  to  the  elementary 
gases  and  pure  water. 


266  BACTERIOLOGICAL  EXAMINATIONS 

In  the  interior  of  closed  tanks  and  in  the  depths  of  sand  filters 
anaerobic  conditions  prevail.  On  beds  of  coke,  and  on  the  surface 
of  sand  filters,  aerobic  conditions  obtain.  The  effluent  from  a 
septic  tank  sewage  disposal  plant  is  very  often  pure  water  from 
both  chemical  and  bacteriological  standpoints,  due  to  the  chemical 
action  of  the  bacteria. 

Bacteriology  of  the  Air. 

That  the  lower  layers  of  the  earth's  atmosphere  contain  many 
bacteria  is  well  known.  The  air  over  the  sea  and  over  mountain 
ranges  is  freer  from  bacteria  than  is  the  air  over  arable  lands  and 
large  cities. 

When  air  is  still  and  confined,  all  bacteria,  according  to  Tyndall, 
gravitate  to  the  ground,  and  the  air  above  becomes  quite  sterile. 
The  atmosphere  of  sick  rooms,  hospitals,  public  conveyances, 
theatres,  etc.,  contains  many  bacteria  and  often  pathogenic  ones. 

The  pus  cocci,  tubercle  bacilli,  and  the  organisms  causing  small- 
pox, scarlet  fever,  and  measles,  all  may  contaminate  the  air. 

The  number  of  bacteria  in  a  given  quantity  of  air  may  be  accu- 
rately measured  by  means  of  a  Sedgwick-Tucker  aerobioscope ;  this 
consists  of  a  large  cylindrical  glass  vessel  opening  at  either  end  into 
various  tubulations.  (Fig.  85.)  Into  one  of  these  granulated 
sugar  may  be  packed;  the  ends  are  then  plugged  with  cotton  and  the 
apparatus  sterilized.  To  examine  the  air,  a  litre  or  more  is  drawn 
through  the  sugar  and  the  latter  is  then  shaken  into  the  large 
cylinder  where  it  is  dissolved  in  melted  gelatine  culture  media. 
The  latter  is  distributed  over  the  interior  of  the  glass  and  allowed 
to  harden.  All  the  bacteria  that  were  in  a  litre  of  air  having  been 
mixed  with  gelatine  and  those  that  are  not  strict  anaerobes  grow  in 
the  gelatine  and  a  number  of  colonies  can  then  be  counted. 

The  dust  of  dwellings  and  streets  contains  most  of  the  bacteria. 
Dried  sputum  is  ground  under  foot  and  swept  up  in  gusts  of  wind, 
and  the  contained  bacteria  are  thus  inhaled  and  do  harm.     The 


BACTERIOLOGY   OF   THE   SOIL 


267 


air  coming  quietly  from  the  lungs  is  pure  and  sterile.  Even  in  active 
disease  processes  of  the  throat  this  is  true.  In  case  the  breath 
comes  violendy,  as  in  speaking,  coughing,  and  sneezing,  the  reverse 
is  the  case.  In  general  it  may  be  put  down  as  an  axiom  that  disease 
germs  cannot  rise  from  a  fluid,  such  as  sewage.  If  they  could  it 
would  mean  that  they  are  lighter  than  air,  which  is  not  the  case. 
Sewer  gas,  as  a  rule,  is  a  bearer  of  some  pathogenic  bacteria  chiefly 
cocci  but  in  reality  it  is  purer  than  generally  supposed.  The 
spread  of  organisms  from  sewage  only  extends  3-6  metres  into  the 
atmosphere  and  then  only  by  the  bursting  of  bubbles  in  the  presence 
of  gas  under  pressure.  This  is  of  course  in  the  absence  of  extra- 
neous air  currents  as  far  as  possible. 


iS 


Fig.  85. — Sedgwick-Tucker  aerobioscope.     (Williams.) 


Bacteriology  of  the  Soil. 

At  least  two  forms  of  pathogenic  bacteria  are  habitually  found  in 
the  soil.  The  tetanus  bacillus,  it  is  well  known,  exists  in  garden 
earth,  manure,  and  top  soil  generally.  Dirt  getting  into  wounds 
is  the  most  frequent  cause  of  tetanus.  Drinking  water  laden  with 
soil  has  been  known  to  have  in  it  tetanus  bacilli,  and  if  used  in 
an  unsterilized  condition  in  wounds  or  when  a  comparatively 
feeble  antiseptic,  such  as  creolin,  has  been  added,  it  may  cause 
tetanus. 

The  bacillus  of  malignant  oedema  has  also  been  isolated  from 
soil.  Streptococci  and  colon  bacilli,  too,  have  been  found  in  garden 
soil.  Typhoid  bacilli  may  contaminate  soil,  but  do  not  multiply 
in  it.  In  sandy  soil  100,000  bacteria  per  gram  have  been  found, 
in  garden  soil  1,500,000  bacteria  per  gram,  and  in  sewage  polluted 
soil  115,000,000  bacteria  per  gram  have  been  determined.     The 


268  BACTERIOLOGICAL  EXAMINATIONS 

first  few  inches  of  ordinary  soil  contain  most  of  the  bacteria,  after 
a  depth  of  two  metres  no  bacteria  at  all  are  found  and  the  earth 
is  sterile. 

Soil  may  be  collected  in  sterile  sharp  pointed  iron  tubes,  and 
diluted  with  sterile  water  of  given  quantity  and  plates  poured 
from  it. 

Arable  lands  may  be  enriched  very  much  by  inoculating  them 
with  certain  nitrifying  bacteria,  some  of  which  convert  ammonia 
into  nitrous  acid,  which  form  in  them  nitrites ;  others  change  nitrites 
into  nitrates  (nitrosomonas).  Certain  of  these  bacteria  are  con- 
cerned in  the  assimilation  of  nitrogen  from  the  atmosphere  and 
adding  to  the  nitrogen  content  of  the  soil,  thus  enriching  it.  On 
the  roots  of  some  plants,  alfalfa,  beans,  peas,  and  clover,  minute 
tubercles  develop.  These  little  growths  are  caused  by  the  nitrify- 
ing bacteria,  and  add  to  the  nutrition  of  the  plant  by  adding  to  it 
ammonia. 

Bacteriology  of  Cow*s  Milk. 

Theoretically  the  milk  in  the  interior  of  the  breasts  of  nursing 
women  and  the  udders  of  cows  is  sterile.  So  soon  as  it  leaves  the 
nipple  it  becomes  contaminated  with  bacteria,  and  by  the  time  it 
reaches  the  pail,  in  the  case  of  cow's  milk,  it  is  far  from  sterile. 

Bacteria  of  the  air,  and  dust  from  the  cattle  and  bedding,  at  every 
movement  of  the  cow,  and  by  the  agency  of  flies,  find  their  way  into 
milk  and  contaminate  it.  The  number  of  bacteria  that  develops 
in  the  milk  depends  upon  the  number  that  reach  it  in  the  first  place, 
the  temperature  of  the  air,  and  the  length  of  time  milk  is  kept  at 
a  temperature  favorable  for  their  multiplication.  Two  hundred 
and  thirty-nine  different  varieties  of  bacteria  have  been  isolated 
from  milk  at  different  times. 

Pathogenic  varieties  of  bacteria  that  are  found  in  cow's  milk 
include  the  tubercle  bacillus,  Streptococcus  pyogenes,  Staphylococ- 
cus aureus,  the  colon  bacillus,  typhoid  bacillus,  the  diphtheria  bacil- 


BACTERIOLOGY   OF   COW'S    MILK  269 

lus,  and  a  whole  host  of  bacteria  that  sour  or  ferment  the  milk  and 
render  it  unwholesome  or  poisonous  for  young  children. 

Cattle  may  be  tuberculous,  and  the  tubercle  bacilli  may  reach 
the  milk  in  this  way.  There  may  be  abscesses  of  the  udder  and  the 
streptococci  from  the  pus  may  cause  infection  in  those  that  use  it. 
Ordinary  follicular  tonsillitis  may  be  caused  in  this  way. 

Bacteria  may  develop  rapidly  in  milk,  which  is  a  good  culture 
medium,  until  they  number  many  million  per  cubic  centimeter 
(sometimes  200,000,000). 

In  good  milk  the  number  of  bacteria  may  increase  when  the  tem- 
perature is  90°  F.,  from  5,200  originally  in  the  milk  immediately 
after  milking,  to  654,000  in  eight  hours. 

By  exposing  milk  to  a  temperature  of  165°  F.  for  twenty  to  thirty 
minutes  and  quickly  cooling  (Pasteurization)  most  of  the  non-spore 
bearing  bacteria  are  destroyed,  so  that  the  number  may  be  reduced 
99.999  percent  by  this  process.  The  pasteurization  of  milk  has 
become  an  economic  problem  of  great  importance  in  large  com- 
munities and  is  not,  as  it  should  be,  sufficiently  supervised.  That 
method  is  best  in  which  milk  is  held  at  146°  F.  for  30  minutes. 
No  harm  is  done  to  the  nutritional  value  of  the  milk.  Some 
authorities  maintain  that  bacteria  grow  no  better  in  pasteurized 
than  in  unheated  milk,  while  others  claim  the  reverse.  More 
evidence  is  on  the  side  of  the  second  view.  The  practical  im- 
portance of  the  controversy  is  that  milk  whether  heated  or 
not  should  be  kept  at  a  temperature  at  which  bacteria  will  not 
multiply,  under  60°  F.  Pasteurized  milk  is  safest  in  time  of 
typhoid  epidemic.^. 

Absolute  cleanliness  on  the  part  of  the  milker,  the  use  of  sterilized 
gloves  and  clothes,  the  absence  of  flies,  dust,  and  the  immediate 
disposal  of  manure,  the  filtration  of  the  milk  after  collection,  the 
immediate  cooling  of  it,  the  uses  of  sterilized  milk  cans  and  bottles, 
all  lessen  the  bacterial  content  of  milk.  It  then  keeps  better,  and  is 
a  wholesomer  and  safer  food  for  infants,  especially  in  hot  weather. 

By  drinking  water  containing  typhoid  bacilli  cows  cannot  be 


270  BACTERIOLOGICAL   EXAMINATIONS 

sources  of  typhoid  infection  through  the  milk.     The  typhoid  bacilli 
are  not  transmitted  through  the  bodies  and  udders  of  the  animals. 

A  bacteriologic  examination  of  milk  comprises  a  total  count, 
the  presence  of  colon  bacilli,  streptococci  in  pus  cells,  tubercle 
bacilli  and  special  species  as  the  case  suggests.  The  first  is  done 
as  given  for  water,  as  is  the  second.  The  discovery  of  streptococci 
is  made  by  centrifugalizing  a  definite  quantity  and  examining  the 
sediment  for  chains,  particularly  in  relation  to  leucocytes,  the  pus 
cells.  Tubercle  bacilli  are  found  by  injecting  guinea  pigs  or  by 
dissolving  the  milk  in  antiformin  (i  part  milk  and  i  part  15  percent 
antiformin)  warming  and  examining  the  sediment  after  centrifu- 
galization, 


NDEX. 


Abscesses,  137 

Achorion  Schoenleinii,  210 

Acid,  benzoic,  51 

boric,  122 

fast,  86 

hydrochloric,  34,  122 

lactic,  128,  149 

production,  113 
Acids,  122 

mineral,  122 
Acne,  137 

Action,  hydrolytic,  22 
Actinomyces,  3 

bovis,  201 

farcinicus,  203 

madura,  204 
Acquired  immunity,  41 
Active  immunity,  41 
Aerobes,  17 
Aerobioscope,  266 
Aerogenes  mucosus,  148 
i^stivo-autumnal  parasites,  226 
Agar- agar,  103 
Agar,  blood,  104 

glycerine,  103 
Agglutinins,  9,  48,  55,  153 
Aggressins,  39 
Air,  bacteria  of,  266 

liquid,  19 
Alcohol,  124 
Alexins,  46,  55 
Allergic,  58 

Alternate  generation,  211,  237 
Amboceptor,  46,  55 
Ammonia,  114 
Amoebae,  211,  212 


Amoeba  dysenteriae,  29,  213 
Amoeboid  motion,  10 
Amphitrichous  bacteria,  9 
Anaerobes,  17 
Anaerobic  culture,  113,  115 
Anaphylaxis,  58,  60 
Aniline  dyes,  85 
Animal  experiments,  117 

parasites,  211 
Anopheles  maculipennis,  227 
Anthrax  bacillus,  14,  29,  31,  45,  88, 
164 

vaccine,  74,  168 
Anti-aggressins,  40 
Anti-bacteriolysins,  62 
Antibody,  45,  56 
Anti-complement,  56 
Anti-ferments,  55 
Antigens,  55,  56 
Anti-immune  body,  56 
Anti-leucocidin,  63 
Anti-plague  serum,  68,  73,  147 
Antiseptics,  120 
Antiseptic  values,  relative,  125 
Anti-toxins,  38,  49,  55 

for  animal  toxins,  63 
Anti- toxin  for  botulism,  63,  68,  178 

for  diphtheria,  63,  64,  191 

for  dysentery,  159 

for  Malta  fever,  68 

for  plant  toxins,  63 

for  pyocyaneus,  63,  68,  163 
staphylococcus,  68 
streptococcus,  67 

for    symptomatic    anthrax,    63, 
176 


271 


272 


INDEX 


Anti-toxin  for  tetanus,  63,  64,  66, 172 

manufacture  of,  63 

standardization  of,  65 
Arethrospores,  14 
Arnold  sterilizer,  98 
Artesian  wells,  264 
Aspergillus  flavus,  209 

fumigatus,  209 

niger,  209 
Attenuation  of  bacteria,  31 
Autoclav,  97 
Autopsies,  animal,  118 
Avenue  of  infection,  33,  151 

Babes  Ernst  granules,  15 

tubercles,  236 
Babesia,  232 
Bacillus,  2,  6 

aerogenes  capsulatus,  88,  1 78 

of  anthrax,  14,  29,  31,  45,  88,  164 

of  blue  pus,  161 

botulinus,  177 

Chauvoei,  174 

of  cholera,  181 

colon,  88,  154,  261,  267 

comma,  181 

of  dysentery,  29,  88 

of  diphtheria,  29,  88,  187 

Friedlander's,  147 

fusiformis,  181 

Gartner's,  160 

of  glanders,  29,  185 

Koch  Weeks,  143 

lepra,  29,  198,  200 

of  lockjaw,  168 

mallei,  29,  88,  185 

of  Malta  fever,  29,  140 

malignant  oedema,  29,  173,  267 

of  malignant  oedema,   29,    173, 
267 

Morax  and  Axenfeld,  143 


Bacillus,of  pseudo-diphtheria,  191,1 

of  plague,  29,  188 

proteus  vulgaris,  30 

pyocyaneus,  88,  161 

rauschbrand,  174 

of  soft  chancre,  163 

smegma,  198 

of  symptomatic  anthrax,  1 74 

of  tuberculosis,  29,  30,  86,  88, 
118,  192,  269,  270 

typhosus,  29,  88,  149,  267 

of  tetanus,  29,  68,  88,  267 

Xerosis,  192 
Bacteria,  attenuation  of,  31 

biological  conditions  of  growth, 

17 
chemical  composition  of,  16 
chromogenic,  21 
definition  of,  i 
disposal  of,  30 
.  fixed  strains  of,  32 
higher,  15 

increasing  malignancy  of,  52 
lophotrichous,  9 
measuring  of,  8 
mesophilic,  18 
of  air,  266 
of  milk,  268 
of  mouth,  34 
of  skin,  34 
of  soil,  267 
of  stomach,  34 
parasitic,  28 
photogenic,  21 
psychrophilic,  18 
reproduction  of,  11 
staining  of,  84 
study  of,  83,  I  OS 
submicroscopic,  30 
thermophilic,  18 
Bacteriaceae,  2,  14 


INDEX 


273 


Bacterial  energy,  21 

proteins,  25 
Bacterins,  137,  139 
Bacteriological  diagnosis,  261 
Bacteriolysins,  9,  39,  55,  56 
Bacteriolysis,  56 
Bacterium,  2 

aerogenes,  148 

Bulgaricum,  149 

coli,  154 

enteriditis,  98 

influenzae,  88,  144 

lactis  aerogenes,  148 

mucosus,  148 

pestis,  88,  144 

pneumoniae,  147 

ulceris  chancrosi,  163 
Balantidium,  212 
Beggiatoa,  4,  9 
Beggiatoaceae,  4 
Benzoate  of  soda,  51 
Benzoic  acid,  51 
Benzol  ring,  50 
Biological   conditions  of  growth  of 

bacteria,  17 
Bismarck  brown,  17 
Black-leg  vaccine,  75 
Blastomycosis,  207 
Bla£,toraycetes,  16 
Blood  agar,  104 

serum,  100,  105 
Blue,  methylene,  86 

pus  bacillus,  161 
Boils,  137 

Bordet-Gengou  bacillus  of  whooping- 
cough,  143 
Botulism,  177 
Bouillon,  loi 
Bovine  tuberculosis,  197 
Bromine,  122 
Bronchitis,  142 


Brownian  motion,  10,  83 

Capsules,  8,  15 
Capsule  staining,  89 
Carbol  fuchsin,  86 

thionin,  88 
Carbolic  acid,  123 
Carbuncles,  137 
Carriers,  82,  134 
Cell  division,  11 
Cellulo-humeral  theory,  45 
Centrosome,  217 
Cercomonas,  212 
Chain  coccus,    127 
Chauvoie,  bacillus  of,  174 
Chemo taxis,  18,  43,  45 
Chlamydobacteriaceae,  3,  7 
Chloride  of  lime,  122 

of  zinc,  1 24 
Chlorine,  122 
Cholera  bacillus,  181 
Cholera,  vaccination  against,  71 
Chromogenic  bacteria,  21 
Ciliata,  212 
Cladothrix,  3 
Classification,  i,  4 
CO2,  21 
Coccaceae,  i 
Cocci,  5 

Coccidia,  212,  231 
Coccidum  hominis,  231 
Coccus  chain,  127 
Coccus,  Malta  fever,  140 

of  meningitis,  132 
Cold,  influence  of,  19 
Coley's  fluid,  79 
Collodion  sac,  100 
Colon  bacillus,  88,  154,  261,  267 
Comma  bacillus,  181 
Complement,  45,  46,  48,  55,  56 

fixation,  60 


274 


INDEX 


Complement,  deviation,  62 
Complementophile,  56 
Conjunctivitis,  139,  142,  143 
Copper  sulphate,  120,  121 
Copula,  55 
Cornybacterium  diphtheriae,  187 

pseudo-diphtheriae,  192 
.Counting  bacteria,  263 
Crenothrix,  4 
Creolin,  123 
Cresol,  123 

Culture  media,  17,  96,  262 
Cultures,  105 

anaerobic,  113,  115 

plate,  108 
Cyclasterion  Scarlatinale,  240 
Cytase,  44,  46,  55 
Cytolysins,  48 
Cytolysis,  48 
Cytophile,  56 
Cytoplasm,  8 

Cytoryctes  variolae,  69,  238 
Cytotoxins,  55 


Direct  division,  84 
Disinfectants,  120 
Drigalski-Conradi  media,  262 
Dum-dum  fever,  218 
Dyes,  aniline,  85 
Dysentery,  amoeba,  213 
bacillus,  157 

Ectosarc,  213 
Egg  cultures,  105 
Ehrlich's  theory,  45,  50 
Endocarditis,  128,  132,  137,  139,  161 
Endosarc,  213 
Endospores,  12 
Endotoxins,  25-39 
Entamoeba  coli,  214 

histolytica,  215 

tetragena,  215 
Enzymes,  22,  59 
Erysipelas,  128 
Esmarch's  method,  112 
Exhaustion  theory,  42 
Experiments,  animal,  117 


Dark  field  illumination,  95 

Darkness,  influence  of,  18 

Dengue  fever,  240 

Desmon,  55 

Diarrhoea,  128,  159 

Differentiation  of  B.   typhosus  and 

B.  coli,  263 
Dilution  method,  107 
Diphtheria,  128,  137 

an ti- toxin,  63,  64,  191 

bacillus,  187 

stain,  93 

toxin,  25,  37 
Diplococcus,  5 

gonorrhoea,  137 

lanceolatus,  129 

meningitis,  88,  132 


Farcin  du  Boeuf,  203 
Favus,  210 
Ferments,  55 

diastic,  22 

tryptic,  22 
Fermentation  tubes,  113 
"Filters,  30,  100,  264 

alum,  264 

Kitasato,  loi 

Pasteur,  265 

sand,  264 
Fixateur,  55 
Fixation,  85 
Flagella,  9,  11 

staining,  91 
Flagellata,  212,  214 
Fomites,  237 


INDEX 


275 


Foot  and  mouth  disease,  241 
Formaldehyde,  123 
Fractional  sterilization,  97 
Friedberger's  theory,  59 
Friedlander's  bacillus,  147 
Fuchsin  solutions,  86 

Ganglia,  235 

Gartner's  bacillus,  160 

Gaseous  edema  bacillus,  178 

Gastric  juice,  34 

Gelatine,  102 

Generation,  alternate,  211,  237 

Giemsa's  stain,  89 

Glanders  bacillus,  185 

Glossina  palpalis,  218 

Gonidia,  11,  13 

Gonococcus,  133,  137 

Gonorrhoea,  139 

Gram's  method  of  staining,  87 

Granules,  chromophilic,  8 

Babes  Ernst,  15 
Gregarinida,  212 
Gregarines,  212 
Gruber-Dunham  reaction,  152 
Gymnamoebida,  212 
Gymnobacteria,  9 

H,  21 
H2S,  21 

Haemolysins,  55 
Haemolysis,  46,  60 
Haemolytic  serum,  46,  47 
Hagmosporidia,  211,  212,  223 
Haffkine,  71,  73 
Halogens,  122 
Hanging  drop,  83 
Haptophores,  52,  56 
Heptotoxin,  55 
Heterotrichida,  212 


Hiss'  capsule  stain,  90 
Histological  methods,  118 
Human  tubercle  bacilli,  197 
Hydrogen  peroxide,  123 
Hydrochloric  acid,  34,  122 
Hydrophobia,  77,  234 
Hyphomycetes,  16,  209 
Hypersusceptibility,  58 

Ice,  bacteria  in,  265 

Immune  body,  47,  53,  55,  56,  58 

Immunkorper,  55 

Immunity,  41,  152 

acquired,  41,  152,  172 

active,  41 

anti-bacterial,  41 

anti- toxic,  41 

inherited,  41 

local,  ss 

natural,  41,  152,  172 

passive,  41 

racial,  41 
Incubator,  99 
Index,  opsonic,  80 
Indol  production,  114 
Infection,  27 

mixed,  45 

phlogistic,  35 

secondary,  32 

terminal,  34 

toxic,  35 

septic,  35 
Infestation,  27 
Influenza  bacillus,  141 
infusoria,  212 
Inoculating  animals,  117 
Inoculating  media,  106 
Insects,  34 

Intermediary  bodies,  55 
Involution  form,  7 
Iodine,  122 


276 


INDEX 


Jenner,  70 
Jenner's  stain,  88 

Kidneys,  excretion  of  bacteria  by,  30 
Kitasato  filter,  loi 
Klebs-LofBer  bacillus,  187 
Koch's  postulates,  29 
Kruse's  scheme,  28 

Lactic  acid,  128,  149 
Laboratory  technique,  96 
Larva  of  mosquitos,  230 
Lateral  chain  theory,  45,  50 
Law  of  multiples,  49 
Leischman's  stain,  88 
Leischman-Donovan  bodies,  218 
Lepra  bacillus,  198,  200 
Leptothrix  Buccalis,  207 

Vaginalis,  207 
Leuococytosis,    44 
Leutin,  26-82 
Lightning  rod  theory,  53 
Lime,  124 

chlorinated,  122 
Local  immunity,  S3 
Lockjaw  bacillus,  168 
LofHer's  blood  serum,  105 

blue,  86 

flagella  stain,  92 

method  of  staining  tissues,  119 
Lophotrichous  bacteria,  9 
Lysis,  47 
Lysol,  123 

Macrogametes,  225,  227 

Macrophages,  44 

Madura  foot,  205 

Malarial  parasites,  223 

Malignant  oedema,  bacillus  of,  173 

Mallein,  26,  77,  186 

Malta  fever,  bacillus  of,  29,  140 


Mannaberg's  scheme,  224 
Mastigophora,  212 
Measles,  241 
Measuring  bacteria,  8 
Meat  poisoning  bacillus,  177 
Membrane,  false,  24 
Meningococcus,  132 
Meningitis,  128,  131,  143,  161 

anti-serum,  134 
Mercury  salts,  120 
Merismopedia,  2 
Merizoites,  225 
Mesophilic  bacteria,  18 
Metals,  influence  of,  20 
Micrococcus,  2,  5 

catarrhalis,  88,  134 

epidermidis  alb  us,  137 

gonorrhoea,  88,  137 

melitensis,  140 

pyogenes,  135 

tetragenus,  139 
Microgametes,  225,  227 
Microgametocytes,  225 
Microphages,  44 
Microspira,  2 
Microsporon  furfur,  210 
Milk,  bacteria  of,  268 

litmus,  103 
Molecule,  toxin,  52 
Monadida,  212 
Monkey  injection,  239 
Monotrichious  bacteria,  9 
Mordants,  86 
Mosquitos,  227 

anopheles,  227 

larva,  230 
Moulds,  16,  209 
Muir-Pitfield  flagella  stain,  91 
Multiplication  of  bacteria,  11 
Mycelia,  16 
Mycobacteriaceae,  3 


INDEX 


277 


Mycobacterium,  3,  7 

lepra,  200 

tuberculosis,  192 
Mycoprotein,  16 
Myxomycetes,  43 

Needles,  inoculating,  ic8 

platinum,  106 
Neisser's  stain,  93 
Negri  bodies,  235 
Nephrotoxin,  55 
Neutralization  of  media,  loi 
Nitrites,  115 

reduction,  22 
Nitrifying  bacteria,  268 
Nitrogen,  22 
Novy  jars,  116 
Nutriment  of  bacteria,  17 

Oidium  albicans,  207 
Oidium  coccidoides,  208 
Oidiumycosis,  207 
Oocysts,  227 
Ookinets,  227 
Opsonins,  55,  80,  132 
Opsonic  index,  80 
Organelles,  212 
Osteomyelitis,  128,  137 

Paracolon  bacillus,  154 
Paratyphoid  bacillus,  154 
Parasites,  animal,  17,  211 
Pasteur  filter,  265 
Pasteurization  of  milk,  269 
Pathogens,  23 

Peptone  solution  (Dunhams),  104 
Pericarditis,  131 
Peritonitis,  128,  131 
Peritrichous  bacteria,  9 
Peroxide  of  hydrogen,  1 23 
Petrie  dishes,  109 


Pfeiffer's  reaction,  46,  47,  48 
Phagocytes,  43 
Phagocytosis,  42,  44,  80 
Phagolysis,  44 
Phlogistic  infection,  35 
Photogenic  bacteria,  21 
Phragmidothrix,  4 
Pink  eye,  143 
Piroplasma  bigemina,  232 
Pitfield's  flagella  stain,  92 
Pityriasis  versicolor,  210 
Placenta,  infection  through,  35 
Plague  bacillus,  146 

vaccination,  73,  147 
Planococcus,  i,  6 
Planosarcina,  i,  6 
Plasmins,  25,  36 
Plasmodium  falciparum,  226 

malariae,  212,  224,  231 

vivex,  225,  231 
Pleomorphism,  7 
Pleuritis,  131,  139 
Pneumococcus,  129 
Pneumonia,  128,  131,  142,  147 
Poliomyelitis,  240 
Polymastigida,  212 
Poly  mites,  225 
Porcelain  filter,  100 
Postulates,  Koch's,  29 
Potato,  104 

Potassium  permanganate,  124 
Pragmidiothrix,  4 
Preparateur,  55 
Proteins,  bacterial,  25 
Precipitins,  49,  55 
Protozoa,  211,  212 

staining  of,  94 
Pseudomonas,  2 
Psychrophilic  bacteria,  18 
Ptomaines,  23,  36 
Puerperal  fever,  128,  137 


278 


INDEX 


Pus,  24,  37 

Pyocyaneus,  anti- toxin,  63,  68,  163 

bacillus,  161 
Pyroplasma  humanis,  233 

Quartan  malarial  parasite,  224 

Racial  immunity,  41 
Rauschbrand  bacillus,  174 
Ravenel  potato  cutter,  104 
Ray  fungus,  201 
Reactivation,  46,  50 
Receptors,  52,  58 
Relapsing  fever,  221 
Retention  theory,  42 
Rheumatic  tetanus,  169,  171 
Rhizopoda,  212 
Ringworm,  210 
Rod  bacteria,  2 
Roll  culture,  1 1 1 
Romanowsky's  stain,  89 
Roux  regulator,  99 

Sac,  collodion,  100 

Saccharomycetes,  Bussi,  208 

Sand-fly  fever,  240 

Sapraemia,  27 

Saprogens,  23 

Saprophytes,  17 

Sarcina,  2,  5 

Sarcode,  212 

Sarcodina,  22 

Scarlatina,  128 

Scarlet  fever,  239 

Schizomycetes,  i 

Schizogony,  213,  227 

Secondary  infections,  32, 128, 137,154 

Septic  infections,  36 

tank,  265 
Septicaemia,  128,  137 

pneumococci,  131 


Serum,  anti-plague,  68,  73,  147 

anti-pneumonococcus,  68 

anti-toxic,  65 

hsemolytic,  60 

reactivated,  46,  50 
Sessile  phagocytes,  43 
Sewage  disposal,  265 
Silver  salts,  122 
Skin,  disinfection  of,  125 
Sleeping  sickness,  217 
Small  pox,  69,  137,  238 
Smegma  bacillus,  198 
Soft  chancre  bacillus,  163 
Soor,  207 

Spermophilus  Columbianus,  233 
Spermo toxin,  55 
Spirillaceae,  2,  181 
Spirillum,  2,  6 

cholera,  88,  181 
Spirochasta,  3,  6,  211,  212 

carteri,  221 

duttoni,  221 

Novi,  221 

obermeieri,  221 

pallida,  219 

refringens,  219,  221 

vincenti,  181 
Spirosoma,  2 
Sporangia,  16 
Spore  staining,  90 
Spores,  II,  98 
Sporoblasts,  227 
Sporogony,  213,  227 
Sporozoa,  212,  223 
Sporozoites,  227,  231 
Sporulation,  12,  84 
Spotted  fever,  232 
Stain,  Bismarck  brown,  87 

Fuchsin  solution,  89 

Giemsa's,  89 

Gram's,  87 


INDEX 


279 


Stain,  Hiss'  capsule,  90    * 

Leischman's,  88 

Loffler's  methylene  blue,  86 
flagella,  92 

Neisser's  diphtheria,  93 

Pitfield's  flagella,  92 
modified  by  Muir,  91 

spore,  90 

thionin  blue,  88 

tubercle  bacilli,  94 

Weigert's,  87 

Wright's,  88 

Welsh's  capsule,  89 

Zeihl's  carbol-fuchsin,  86 
Staining  bacteria,  84,  85 
Standardization  of  anti-toxins,  65 
Staphylococcus,  2,  5,  80,  81 

albus,  135 

aureus,  30,  135,  141 

citreus,  135 

pyogenes,  8S 
Stegomyia  Fasciata,  237 
Sterilization,  96 

culture  media,  97 

fractional,  97 

glassware,  96 
Sterilizer,  Arnold,  98 
Stomach,  bacteria  of,  34 
Street  virus,  235 
Streptococcus,  2,  32,  267 

anti- toxin,  67 

erysipelas,  79 

intraceUularis,  132 

lanceolatus,  6,  129 

mucosus,  132 

pneumoniae,  88 

pyogenes,  88,  127 

viridans,  132 
Strep  to  thrix,  3 

hominis,  205     ^ 

madura,  204 


Study  of  bacteria,  105 

Substance  sensibilisatrice,  55 

Suctoria,  221 

Sulphur  dioxide,  1 24 

Symptomatic  anthrax  anti-toxin,i76 

bacillus,  174 
Symbiosis,  18 
Syphilis,  221 

Table  of  characteristics  of  bacteria, 

242 
Temperature,  influence  on  growth,  18 
Terminal  infection,  34 
Tertian  fever,  225 
Test,  tuberculin,  77 
Tetanolysin,  38 
Tetanospasmin,  38 
Tetanus  anti- toxin,  63,  64,  66,  172 

bacillus,  29,  68,  88,  267 

rheumatic,  168,  171 

spore,  37 

toxin,  26,  38,  45 
Tetrads,  5 

Theory,  cellulo-humeral,  45 
Thermolabile,  46 
Thermostat,  99 
Thionin,  88 
Thio  thrix,  4 

Thrombosis  formation,  24 
Thrush,  207 
Tonsillitis,  128 
Toxalbumins,  37 
Toxic  infection,  36 
Toxin,  24,  36,  55 

molecule,  50 
Toxoid,  38,  53 
Toxons,  38 
Toxophores,  52,  56 
Trachoma,  241 
Treponema,  212 

pallida,  82,  219 


28o 


INDEX 


Trichobacteria,  9 
Trichomonas,  212 
Trichophyton,  210 
Trypanoma,  211,  212 

brucei,  215,  218 

cruzi,  218 

equiperdum,  216 

evansii,  216,  218 

gambiense,  215,  217 

lewisi,  216,  218 

nocturna,  216 
Tsetse  fly,  215,  218 
Tubercle  bacillus,  198 

stain,  94,  199 
Tubercles,  Babes,  236 
Tuberculin,  25,  76,  80,  198 

T.R.,  76 
Tuberculosis,  128 
Turpentine,  124 
Tyndallization,  97 
Typhoid  bacilli,  29,  88,  149,  267 

in  water,  261 

vaccination  against,  72 
Typhus  fever,  240 

Udder,  infection  by,  261 
Unit  of  anti-toxin,  65 

toxin,   65 
Uterus,  bacteria  in  normal,  35 


Vaccinia,  69,  238 
Variola,  70,  238 
Venom,  52 
Virus  fixe,  71,  235 
Vibrio,  2,  6 

cholera,  181 

Metchnikovii,  185 

protens,  185 

Schulykilliensis,  185 

septique,  173 

tyrogenum,  185 
Vincent's  angina,  181 
Virulency,  31 

Wassermann's  list  of  anti- toxins,  63 

test,  61 
Water,  bacteria  of,  261 
Weigert's  aniline  gentian  violet,  87 

method  of  staining  tissue,  119 

theory,  52 
Welch's  capsule  stain,  89 

theory,  62 
Wells,  artesian,  264 
Widal  reaction,  48,  152 
Woelffhiigle  plate,  263 
Wright,  80 
Wright's  stain,  88 

Xerosis  baciUi,  192 


Vacuoles,  8 
Vaccination,  69 

for  plague,  68 
Vaccine,  anthrax,  74,  168 

black  leg,  75 

cholera,  71 

plague,  68,  ys,  i47 

small  pox,  69 

tuberculosis,  75 

typhoid,  68,  72 


Yeasts,  16 
Yellow  fever,  236 

Zeihl's  solution,  86 
Zinc  chloride,  124 
Zooglia,  9 
Zymase,  22 

Zymogenic  bacteria,  21 
Zwischenkorper,  55 
Zymophore,  56 


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